WO2006061992A1 - Preventive/therapeutic composition for liver disease - Google Patents

Preventive/therapeutic composition for liver disease Download PDF

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Publication number
WO2006061992A1
WO2006061992A1 PCT/JP2005/021466 JP2005021466W WO2006061992A1 WO 2006061992 A1 WO2006061992 A1 WO 2006061992A1 JP 2005021466 W JP2005021466 W JP 2005021466W WO 2006061992 A1 WO2006061992 A1 WO 2006061992A1
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WO
WIPO (PCT)
Prior art keywords
composition
insulin
salt
aspartic acid
liver
Prior art date
Application number
PCT/JP2005/021466
Other languages
French (fr)
Japanese (ja)
Inventor
Mihoko Tsuji
Akira Mitsui
Akira Okano
Original Assignee
Ajinomoto Co., Inc.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Ajinomoto Co., Inc. filed Critical Ajinomoto Co., Inc.
Priority to JP2006547795A priority Critical patent/JPWO2006061992A1/en
Priority to EP05809495A priority patent/EP1829540A4/en
Publication of WO2006061992A1 publication Critical patent/WO2006061992A1/en
Priority to US11/759,287 priority patent/US20080038321A1/en

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Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • A61K31/195—Carboxylic acids, e.g. valproic acid having an amino group
    • A61K31/197—Carboxylic acids, e.g. valproic acid having an amino group the amino and the carboxyl groups being attached to the same acyclic carbon chain, e.g. gamma-aminobutyric acid [GABA], beta-alanine, epsilon-aminocaproic acid or pantothenic acid
    • A61K31/198—Alpha-amino acids, e.g. alanine or edetic acid [EDTA]
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/17—Amino acids, peptides or proteins
    • A23L33/175—Amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/4164—1,3-Diazoles
    • A61K31/4172—Imidazole-alkanecarboxylic acids, e.g. histidine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00

Definitions

  • the present invention relates to a composition for preventing and / or treating liver diseases.
  • the present invention also relates to a composition for preventing or treating insulin-related liver disease Z or treatment.
  • N AFLD nonalcoholic fatty liver
  • NASH nonalcoholic steatohepatitis
  • Non-patent Document 6 Insulin resistance is considered to be poor in the pathology of NAFLD / NASH (Non-patent Document 6), and mice that have specifically destroyed the insulin receptor in the liver are insulin resistant, impaired glucose tolerance, and hyperinsulinemia. It has been reported that liver function abnormalities are observed (Non-patent Document 7). In addition, the following reports indicate that insulin resistance is related to the TRB3, LAR, and glucose-6-phosphatase (G6Pase) genes.
  • Non-patent Document 8 the expression of a gene product called TRB3 that suppresses AKT phosphate downstream of the insulin signal is increased in obese and hepatic insulin resistant db / db mice.
  • LAR one of the protein-synthetic phosphatases that suppress insulin signals
  • Non-patent Document 12 a composition that directly or indirectly suppresses expression of genes such as LAR, TRB3, or G6Pase in hepatocytes in vitro or in vivo improves insulin resistance by increasing liver insulin sensitivity.
  • genes such as LAR, TRB3, or G6Pase in hepatocytes in vitro or in vivo improves insulin resistance by increasing liver insulin sensitivity.
  • it may be useful for the prevention and treatment of liver diseases such as NAFLD / NASH.
  • Non-patent Document 2 Thiazolidine-based compounds (pioglitazone, rosiglitazone) are currently approved as therapeutic agents for type 2 diabetes as drugs that improve insulin resistance, and these are effective for various metabolic syndrome symptoms. There is a report. In addition, the results of reconnaissance clinical trials suggesting that these may be effective for liver diseases related to insulin resistance such as NAFLD / NASH have also been reported (Non-patent Documents 2 and 3). However, the main mechanism of action of these drugs is the effect of ppAR y activity on adipocytes, and as a side effect resulting from this, weight gain continues not only during the administration period but also after the end of administration, and liver damage relapses. It is considered a problem (Non-Patent Document 2).
  • Non-patent Document 3 reports of histologically increased inflammation in the portal vein area
  • PPA activity in the liver rather causes fatty liver. From these reports (Non-Patent Documents 4 and 5), the safety of long-term administration of thiazolidine compounds to patients with liver diseases has not been fully confirmed. In other words, long-term preventive and therapeutic effects on liver diseases related to insulin resistance such as NAFLD / NASH are high! Drugs and safe treatment methods are still established!
  • amino acids are highly safe compounds that have been known for a long time, and there have been many nutritional reports on infusions containing amino acids, but NAFLD / NASH related to metabolic syndrome There are few reports of pharmacological effects related to such liver diseases.
  • aspartic acid one of the amino acids
  • MUT metabolic uncoupling therapy
  • Patent Document 2 a physiologically active composition having an antitumor activity, an antihyperlipidemic activity, a blood pressure lowering activity, and a blood glucose lowering activity, comprising an amino acid containing aspartic acid and a plurality of components derived from a crude drug rich in ⁇ -dalcan. Have been filed and disclosed.
  • Patent Document 3 a compound that exhibits an antagonistic action on the incorporation of 3-gua-dinopropionic acid into synaptosomes may have a hypoglycemic action, and thus may be effective in diabetes and obesity.
  • the force DL-Asp in which 119 to 128 potential compounds are disclosed, is also listed in this table.
  • these patent documents have no reports showing that L-Asp alone improves hepatic insulin resistance alone, in addition to those that show an effect on liver diseases.
  • Non-patent documents 13-15 describe the treatment of liver diseases with regard to the salt compound of aspartic acid and other amino acids, but V, the gap is also related to the liver disease related to insulin resistance. There is no description about the effect of.
  • Non-patent document 16 is a study on the short-term accumulation of labeled fatty acids in the liver. Whether or not it exhibits a preventive / therapeutic effect on liver disease related to aspartic acid S insulin resistance. Is shown! /
  • Patent Document 1 US2004 / 0043013 A1
  • Patent Document 2 JP 2003-212775
  • Patent Document 3 Japanese Translation of Heisei 6-510760
  • Non-Patent Document 1 Gastroenterology, 2002; 123: 1702-1704, 1705-1725
  • Non-Patent Document 2 Hepatology, 2004; 39: 188-196
  • Non-Patent Document 3 Hepatology, 2003; 38: 1008-1017
  • Non-Patent Document 4 J. Lipid Res., 2002; 43: 1809-1817
  • Non-Patent Document 5 J. Clin. Invest., 2000; 106: 1221-1228
  • Non-Patent Document 6 Semin. Liver Dis., 2004; 24: 3-20
  • Non-Patent Document 7 Mol Cell., 2000; 6: 87-97
  • Non Patent Literature 8 Science, 2003; 300: 1574-1577
  • Non-Patent Document 9 Diabetes, 2000; 49: 810-819.
  • Non-Patent Document 10 J. Biol. Chem., 1998; 273: 31615-31620
  • Non-Patent Document 11 Diabetes, 1999; 48: 1579-1585
  • Non-Patent Document 12 Am J Physiol Endocrinol Metab., 2004 Nov 2 [Epub ahead of print]
  • Non-Patent Document 13 Fortigee der Medizin (GERMANY), 1996; 114: 141-146,
  • Non-Patent Document 14 Zeitschrift for interntechnik (GERMANY, WEST), 1973; 49: 469
  • Non-Patent Document 15 France europeenne en toxicologie (FRANCE), 1978; 1: 303-309
  • Non-Patent Document 16 Therapie (FRANCE), 1966; 21: 719-731
  • An object of the present invention is to provide a composition effective for the prevention and treatment of liver diseases.
  • Another object of the present invention is to provide a food containing the composition for preventing and / or treating liver diseases.
  • Another object of the present invention is to provide a composition for preventing and / or treating insulin resistance related diseases, or a functional food containing them.
  • Another object of the present invention is to provide an insulin sensitivity enhancer and an insulin resistance improver.
  • the present invention has a certain amino acid has an effect of enhancing insulin sensitivity, and also has an action of suppressing the expression of at least one of liver TRB3 or LAR. It has been made based on the knowledge that such amino acids are effective in the prevention and treatment of liver diseases.
  • the present invention provides a composition for the prevention and Z or treatment of liver diseases comprising an amino acid having an insulin sensitivity enhancing action as an active ingredient.
  • the present invention also comprises at least one amino acid selected from aspartic acid, alanine, cystine, glutamine, glycine, histidine, and their salt strength, and a composition for preventing or treating liver disease, which is characterized in that Offer things.
  • the present invention also provides a composition for preventing and treating liver disease or treatment comprising L-aspartic acid and Z or a salt thereof as a single active ingredient.
  • the present invention also includes L-aspartic acid and Z or a salt thereof as an active ingredient, and as a second active ingredient 1) alanine, cystine, glutamine, glycine, histidine and a salt thereof, or 2) other than an amino acid and a salt thereof
  • a liver disease prevention and Z or treatment composition containing at least one of the following liver disease prevention / treatment drugs, or a combination of these liver disease prevention and Z or treatment kits is provided.
  • the present invention also provides a food containing the composition for preventing and treating liver disease or Z.
  • the present invention also provides an insulin resistance-related disease prevention and Z or treatment composition comprising an amino acid having an insulin sensitivity enhancing action as an active ingredient, or a food containing the same.
  • the present invention also provides an insulin sensitivity enhancer or an insulin resistance improver comprising at least one selected from aspartic acid, alanine, cystine, glutamine, glycine, histidine, and the group power of their salt strength.
  • Insulin action in the present invention means that insulin exerts metabolic regulation ability in body tissues. More specifically, insulin binds to insulin receptors of insulin-sensitive cells such as liver, muscle, and adipose tissue, and these cells take up glucose into cells, promote energy utilization and storage, and promote protein synthesis. In general, it refers to cell biology such as cell proliferation. For example, the suppression of hepatic gluconeogenesis by suppressing G6Pase expression in vitro and the decrease in physiological blood glucose level in vivo via these are insulin actions.
  • insulin resistance refers to “decrease in insulin sensitivity”. More specifically and cell biologically speaking, a decrease in the number of insulin receptors, presence of an insulin antagonist, other cells mediated by insulin receptors A state of reduced information transmission ability.
  • LAR which dephosphorylates the substrate phosphate, which is the first step in insulin signaling
  • TRB which also inhibits AKT phosphate, which is essential for a part of the insulin signaling process
  • An increase in expression of 3 implies the presence of insulin resistance at the cellular level (decreased insulin sensitivity).
  • insulin resistance and “decrease in insulin sensitivity” in the present invention are physiological (in vivo) conditions in which an insulin action (decrease in blood glucose level) corresponding to the insulin concentration in blood cannot be obtained.
  • HOMA-IR which is proportional to the product of fasting blood insulin level and fasting blood glucose level
  • QUICKI are used as simple indicators.
  • a more detailed and rigorous evaluation can be performed by combining a glucose tolerance test, an insulin tolerance test, a glucose clamp method, a minimal model method, and the like. For example, even in pathological animals, an increase in the product of fasting blood insulin level and fasting blood glucose level, and high insulin secretion during glucose load, decreased Z glucose tolerance means the presence of insulin resistance (decrease in insulin sensitivity).
  • the "insulin sensitivity enhancing action” may be based on any mechanism as long as it is an action that enhances insulin sensitivity. Anything that enhances the function of the above is acceptable. It may be combined with the action of substituting all or part of the insulin action in the absence of insulin, and supplementing Z or insulin supply, ultimately improving the insufficiency of insulin action in vivo It only has to be an action. That is, the “insulin sensitivity enhancing action” also has the effect of improving “insulin resistance”.
  • “metabolic syndrome” in the narrow sense is the following Table A, published in 2001 by the National Cholesterol Education Program (NCEP) Adult Treatment Panel (ATP). Refers to patients with diseases that meet the diagnostic criteria guidelines (NCEP-IV).
  • the “metabolic syndrome-related disease” in the present invention is not limited to the above-mentioned risk factors and standard values of wrinkles according to the WHO proposal as an integrated concept such as syndrome X, insulin resistance syndrome, multiple risk factor syndrome, etc. (Visceral fat accumulation), insulin resistance (hyperinsulinemia) and related type 2 diabetes (abnormal glucose tolerance), abnormal lipid metabolism (hypertriglyceridemia, VLDL-triglyceride elevation, HDL-cholesterol decline), It is used as a comprehensive name for diseases and symptoms that are thought to be based on common molecular pathologies such as fatty liver, hypertension, and urinary microalbumin.
  • diseases and symptoms that are thought to be based on common molecular pathologies such as fatty liver, hypertension, and urinary microalbumin.
  • non-alcoholic fatty liver (NAFLD) Z non-alcoholic steatohepatitis (NASH) refers to, for example, a statement and technical review published by the American Society of Gastroenterology and the American Society of Liver Disease in 2002. As presented in Non-Patent Document 1), it is diagnosed with fatty liver, inflammation, and fibrosis (Grading. Staging), and it is a disease associated with obesity, diabetes, hyperlipidemia, and insulin resistance.
  • insulin resistance-related liver disease refers to a liver disease in which insulin resistance is mainly involved in its onset or pathogenesis! ⁇
  • NASH non-alcoholic fatty liver
  • NASH nonalcoholic steatohepatitis
  • the “insulin resistance-related disease” in the present invention is a liver disease mainly related to insulin resistance in its onset or pathogenesis, such as metabolic syndrome and metabolic syndrome-related diseases. including.
  • “disease prevention and Z or treatment” means that the above-mentioned disease is not diagnosed, that the person diagnosed with the above-mentioned disease deviates from the definition, and abnormality in the measured value diagnosed with the above-mentioned disease is improved.
  • exacerbation of the abnormality diagnosed as the above-mentioned disease means delaying the progress, for example, maintaining or improving fasting blood glucose level, fasting insulin level, glucose tolerance, liver fat mass.
  • An amino acid having an insulin-sensitizing effect is obtained by culturing cells, for example, rat liver cancer-derived cell line Fao in a medium supplemented with one or several amino acids for 30 minutes to 1 week, more preferably 3 hours.
  • Fao cells can be obtained as ACC No. 89042701 in European and ollection of Animals and ultures (Porton Down, UK);
  • cells such as rat liver cancer cell line Fao are cultured in a medium supplemented with one or several amino acids for 30 minutes to 1 week, more preferably 6 hours.
  • the insulin-like action and the insulin action-enhancing action can be confirmed by, for example, comparative measurement with cells cultured in a medium containing no amino acid, with suppression of the expression level of G6Pase as an index.
  • a specific amino acid actually enhances insulin action can be determined by applying a specific amino acid from 1 day to 1 year, preferably from 3 to 1 for patients or pathological animals lacking insulin action, such as GK rats. Administered for 8 weeks and administered amino acids with indicators of insulin action such as fasting blood glucose, decreased fasting insulin, improved glucose tolerance by oral glucose tolerance test, and decreased expression of TRB3, LAR, G6Pase genes in liver This can be confirmed by comparison with the group that does not.
  • a specific amino acid is administered for 1 to 1 year, preferably 3 to 6 weeks, to a fatty liver patient or a diseased animal prepared by giving a high fat diet, and hepatic triglyceride content, and adipose-related enzyme gene in the liver (For example, malic enzyme (Malic Enzyme), stearoyl CoA desaturase 1 (SCD1)), fat burning system ( ⁇ oxidation) regulation-related transcription factor, enzyme gene (PPAR a, acyl CoA oxidase (ACO), cal-tin palmitoyltransferase) 1 (CPT1)), or the expression of the hepatocyte-specific transcription factor gene (hepatocyte nuclear factor 4a (HNF4a)) related to lipid metabolism control, etc. Can be confirmed.
  • Metabolic syndrome is improved.
  • a specific amino acid is administered to Drome-like pathological animals for 1 to 1 year, preferably 3 to 8 weeks, and no amino acid is administered using abnormal metabolic criteria items such as fasting blood glucose and triglycerides as indicators. This can be confirmed by comparative measurement with the group.
  • Metabolic syndrome-like animals with reduced fatty liver and insulin sensitivity may be normal or diseased animals such as GK rats with sucrose, fructose, or high-fat diet for 1 to 1 year, preferably 2 to 8 weeks. Can be created by administration and loading.
  • the preventive effect can be confirmed by administering a specific amino acid at the same time as a high fat diet or by oral administration, as well as the therapeutic effect.
  • the amino acid used in the present invention may be any pharmaceutically acceptable salt, regardless of whether it exists in nature or not.
  • one or more amino acids from which group power consisting of aspartic acid, alanine, cystine, dartamine, glycine, histidine and their salts are also selected are mentioned.
  • the body is preferred.
  • L-aspartic acid is particularly preferred.
  • an alkali metal salt or alkaline earth metal salt of L-aspartic acid for example, sodium L-aspartate is preferred.
  • composition of the present invention can contain only the above amino acids and Z or a salt thereof as amino acids, but can contain amino acids other than those described above.
  • the amount is preferably 50% by weight or less of the whole amino acid.
  • composition of the present invention may contain various pharmacologically acceptable pharmaceutical substances as a pharmaceutically acceptable carrier (adjuvant) in addition to the above components.
  • composition of the present invention can be prepared in various dosage forms such as oral administration, intraperitoneal administration, transdermal administration, and inhalation administration.
  • suitable solid or liquid preparation forms such as granules, powders, coated tablets, tablets, (micro) capsules, suppositories, syrups, juices, suspensions, emulsions, drops, Examples include infusion solutions, injection solutions, and preparations that prolong the release of active substances.
  • the intake of amino acids used in the present invention is appropriately selected according to the symptom.
  • the daily dose is expressed in terms of the net weight of amino acids in an adult patient per day. More than 1 lg, preferably about 3 g to 300 g, more preferably about 3 to 20 g. In severe cases, the dose can be further increased.
  • the frequency and timing of administration it can be applied once every few days or once a day. Usually, it is administered several times a day, for example, 2-4 times.
  • the above amino acids can be used alone or in combination with other drugs.
  • drugs to be combined include other amino acids and liver disease prevention / treatment drugs.
  • preferred examples of other amino acids include alanine, cystine, glutamine, glycine, histidine, and salts thereof, and alanine is particularly preferred.
  • other preventive and therapeutic agents for liver disease include hepatoprot ectant such as ursodeoxycholic acid, glycyrrhizin, and betaine, as well as preventive and therapeutic effects on liver disease.
  • Antidiabetic drugs such as pioglitazone, rosiglitazone, metformin, clofibrate, gemfib mouth gill, atto mouth bastatin, mouth sultan, probucol, icosapentate ethyl, phosphatidylcholine, polyphosphatidylcholine and other circulatory disease drugs, sibutramine, Preferred are anti-obesity drugs such as orlistat, antioxidants such as vitamin C and vitamin E, and anti-inflammatory drugs such as pentoxifylline and anti-TNF antibody.
  • amino acids can be used in a form combined with a therapeutic agent for other diseases related to metabolic syndrome and insulin resistance, such as nateglinide, which is a rapid-acting insulin secretagogue for diabetes.
  • the combination of the above drugs may be in a form containing both of them in one pharmaceutical composition or in a form in which two pharmaceutical compositions containing each component are administered simultaneously or at some interval.
  • Yo When combined with other amino acids, precursors such as peptides may be used, which decompose in vivo to become these amino acids.
  • the amount of other drugs in the composition can be used in an appropriate amount or dosage as an effective amount of each drug.
  • L-aspartic acid is 20 mg-50 gZ day, preferably 0.1-40 gZ day, more preferably 3-10 g once a day 1-4 times a day.
  • Oral administration is desirable.
  • L-alanine should be administered orally at 5-50 gZ days, more preferably 6 g at a time, 1-3 times a day.
  • the preventive and Z or therapeutic composition of the present invention may be contained in a food, a slurry, a paste, or a milky liquid liquid for medical use, and the use or efficacy described in the package.
  • Examples of the knockout include a so-called Noh book that describes the amino acids contained in a certain amount or more and z or explanations regarding its use, efficacy, and eating and drinking method.
  • Applications' Indications include metabolic syndrome, metabolic syndrome, insulin resistance, liver disease, liver dysfunction, fatty liver, NAFLD, NASH, weight gain, obesity, abdominal obesity, visceral fat accumulation, visceral fat accumulation, diabetes, Impaired glucose tolerance, hypertension, dyslipidemia (hyperlipidemia), hyperinsulinemia, arteriosclerosis, cardiovascular events and the prevention of related symptoms, or clinical indicators and biochemistry related to these This can prevent or improve the inspection value.
  • the content of amino acids in the case of food is 0.1 to 100% by weight, preferably 3% by weight or more, more preferably 6 to LOO% by weight, based on the whole food.
  • foods having an amino acid content of 70 to: LOO weight% that is, foods (functional foods, supplements, etc.) whose main component is only the amino acid of the present invention are also preferred.
  • the content of amino acids is also preferred 3-6 wt 0/0.
  • the content of amino acids can be arbitrarily set so as to achieve the effects of the present invention, depending on the form of food and the daily intake.
  • the ability to use only the amino acids used in the present invention as these amino acids can be used in combination with other amino acids (referred to as other amino acids).
  • other amino acids when other amino acids are used in combination, it is preferably 50% by weight or less of the total amino acids.
  • the amino acid used in the present invention is preferably the amino acid itself, but is not limited to the amino acid itself.
  • the above-mentioned L-aspartic acid is subjected to hydrolysis reaction, particularly L-asparagine by in vivo hydrolysis reaction.
  • a substance capable of becoming an acid for example, a protein or peptide having L-aspartic acid as a structural unit may be used.
  • the foods targeted by the present invention include functional foods containing emulsifiers and fragrances that do not exhibit the above-mentioned uses.
  • V ⁇ L-aspartic acid or a salt alone supplement that exhibits the above-mentioned effects and does not contain other active ingredients as a food subject to the present invention, for example, the functionality of L-aspartic acid alone
  • a composition in the form of a product that can usually ingest lg or more, preferably 3 g or more, more preferably 3 to 40 g per day for an adult is preferable.
  • L-aspartic acid contains more than a certain amount, for example, more than 2 g of L-aspartic acid per unit of intake per meal.
  • examples include, but are not necessarily limited to, concentrated liquid foods for oral use in which the amount of protein other than acids and amino acids is 10 g or less per meal.
  • a liver disease prevention and Z or therapeutic composition comprising an amino acid having an insulin sensitivity enhancing action as an active ingredient, particularly prevention of nonalcoholic fatty liver or nonalcoholic steatohepatitis and A therapeutically effective composition. Furthermore, aspartate, alanine, cystine, glutamine, glycine, and histidine can suppress the expression of TRB3, which is related to liver insulin resistance. It has the advantages of suppressing expression, improving insulin resistance, improving liver fat accumulation in diseased animals, and having no side effects such as weight gain.
  • Fao cells 2xl0 5 cells / well are seeded on a 24-well plate, cultured in RPMI 1640 medium (Nacalai Tester Code No. 30264-85) containing 10% fetal calf serum, 1% culture at 37 ° CCO 5%, then the following
  • the cells were further cultured for 3 hours under% conditions.
  • the composition of the minus amino acid medium is shown in Table 1.
  • the normal medium is a medium obtained by adding the amino acids listed in Table 2 to a minus amino acid medium.
  • fetal bovine serum 100 Adjust pH to 7.2 and sterilize by filtration using 0.22um filter
  • l_Valine 20 Adjust pH to 7.2 and sterilize by filtration using 0.22um filter.
  • TRB3 expression was significantly inhibited by aspartic acid, cystine (Cyt), glutamine, glycine, and histidine (p ⁇ 0.05) (Table 3). There is no significant inhibition of TRB3 expression for arginine, asparagine, dartamic acid, hydroxyproline, isoleucine, leucine, lysine, methionine, phenenolealanin, proline, serine, threonine, tryptophan, tyrosine, norrin, and cysteine. I helped.
  • L-aspartic acid significantly released TRB3 even when added to 0.03 mM to 0.3 mM. Suppressed the present. Betaine does not show an inhibitory effect even when ImM is added, and ortine is 0.3- or higher. When LmM is added, the inhibitory effect is 0.03-0.06 mM, which is the same as that of L-vaspartic acid. It was weaker than acid.
  • Minus amino acid medium 1.000 ⁇ 0.01
  • SEQ ID Nos: SEQ ID Nos: 1-4 were used.
  • Fao cells were seeded on a 24-well plate, cultured for 1 cm, and then RPMI1640 medium (without dalcose, containing 0.1% BSA) excluding L-Asp was basically 0, 0.3, l.OmM L-Asp and 0, 10 - ⁇ , 10- 8, were cultured for 6 hours ⁇ Ka ⁇ the 10- 6 ⁇ insulin. Thereafter, RNA was extracted from the cells, and the expression level of G6Pase was measured by quantitative PCR.
  • Insulin suppresses the ability to increase G6Pase expression in hepatocytes by glucose starvation.
  • L-Asp showed the effect of decreasing the expression of G6Pase even in the culture with addition of single, and enhanced the decrease of G6Pase expression by coexisting with insulin.
  • L-Asp was confirmed to enhance the insulin sensitivity of hepatocytes (Table 4).
  • the G6Pase expression level in Insulin OM, -Asp RPMI is set to 1.00.
  • SEQ ID NO: SEQ ID No: 1, 2, 5, 6 was used.
  • L-Asp The effect of L-Asp on the sensitivity of insulin observed in cultured cell lines in vitro shows sufficient effects in in vivo disease animals.
  • GK rat a diabetic model rat with insufficient insulin secretion Z
  • L-Asp was administered for a long time with diet, and the effect on liver gene expression and disease state was examined.
  • Male GK rats were purchased at 6 weeks of age, and after 1 week pre-breeding, 1 group was divided into 5 groups per group, switched to the experimental diet and bred for 8 weeks.
  • 3AS group 3% L-Asp containing diet
  • 6AS group 6% L-Asp containing diet
  • Fasting blood glucose at the 6th week after administration decreased depending on the dose of L-Asp, and the 6AS group showed a significantly lower value than the CON group. Fasting insulin levels were significantly lower in the 3AS group than in the CON group and decreased in the 6AS group. From these results, it was considered that insulin resistance in GK rats was improved by L-Asp administration.
  • Fasting blood glucose is generally considered to be an index that reflects basal insulin secretion and insulin resistance, and it has a long-term ability to administer Asp. It shows an improvement in insulin basal secretion and / or an increase in insulin sensitivity that reflects liver gene expression. It was thought that it promoted a decrease in blood sugar level.
  • L-Asp modifies gene expression and enhances insulin sensitivity in the liver, as in vitro, for long-term administration to diseased animals.
  • mRNA level is 1.00 for CON group
  • SEQ ID Nos: SEQ ID Nos: 1-8 were used.
  • Example 4 Insulin resistance improving effect and fatty liver improving effect by long-term administration of L-Asp on diseased rats
  • GK rats fed a high-fat diet are considered to be a major factor in the metabolic disorders related to insulin resistance in recent years, such as diabetes and increased fatty liver.
  • the pharmacological action of L-Asp was confirmed.
  • vitamin mix (AIN93) 100 100 100 100 100 100 100
  • the fat content in the liver was found to be 39.9 mg per lg in the HFD group compared to the SD group (17.1 mg).
  • the triglyceride in the liver decreased to 28.0 mg / lg of liver, and the effect was stronger than that of pioglitazone (Table 8). Histological examination also showed improvement in lipid droplet accumulation mainly in the hepatic portal vein area, indicating the effectiveness of L-Asp for fatty liver and NASH.
  • HFD + Pio group showed a significant increase in body weight compared to the HFD group, but the HFD + Asp-administered group had the same body weight transition as the HFD group (Table 8).
  • mRNA level is 1.00 for the SD group
  • SEQ ID Nos: SEQ ID Nos: 1 to 6 and 9 to 14 were used.
  • L-Asp enhances insulin sensitivity and suppresses gluconeogenesis not only in vitro but also in in vivo pathological models that combine insulin secretion failure and insulin resistance. It was confirmed that the effect of improving fatty liver by ingesting a high-fat diet showed an effect.
  • Example 5 Inhibition of L-Asp Insulin Resistance Related Gene (TRB3) Expression and Effect of Addition of Other Amino Acids on Hepatic Lipid Metabolism Related Gene (HNF4 a) Expression Enhancement)
  • Fao cells (2 ⁇ 105 cells / well) were seeded on a 24-well plate and cultured for 1% at 37 ° C. and C02 5% in RPMI 1640 medium (Nacalai Tester Code No. 30264-85) containing 10% fetal calf serum. The next day, RPMI medium (minus amino acid medium, Table 1) with all amino acids removed, or this The medium was replaced with a medium supplemented with L-Asp (concentration 0.05 mM) or L-Ala (concentration 0.03 mM), or both, and the cells were further cultured at 37 ° C, C02 5% for 3 hours.
  • RPMI 1640 medium Nacalai Tester Code No. 30264-85
  • HNF4 a 1.00 1.30 0.80 1.84
  • TRB3 mRNA The expression level of TRB3 mRNA was suppressed to 54% or 43% when L-Asp or L-Ala was added to the medium, and when both L-Asp and L-Ala were added. More strongly, suppression of expression (26%) was observed (26%).
  • HNF4 a mRNA did not increase when L-Ala was added, but increased to 1.3-fold when L-Asp was added, and both L-Asp and L-Ala were added. When drunk, it was much stronger than L-Asp alone, with an increased expression (1.84 times).
  • SEQ ID Nos. 3, 4, 15 and 16 were used.
  • the primer sequences used for gene expression measurement are shown in Tables 11 and 12.
  • Rat iS-Acti n-3442F ⁇ -Act n F SEQ ID NO 1 CTCCAAGTATCCACGGCATAG
  • Rat iS-Acti ⁇ -Act n R SEQ ID NO 2 AAGCAATGCTGTCACCTTCC rTRB3 F TRB3 F SEQ ID NO 3 GTTACCACAGTGCCACATGC rTRB3 TRB3 R SEQ ID NO 4 CAGAAGCCTAGCACCAGACC
  • L-Asp The pharmacological action of L-Asp was confirmed using Wistar rats fed a high fat diet.
  • a high fat diet (containing 30% beef tallow; hereinafter abbreviated as HFD) and an experimental diet with about 6% L-Asp added to HFD were prepared as shown in Table 7.
  • n 6 average standard deviation

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Abstract

A preventive and/or therapeutic composition for liver diseases, comprising as an active ingredient an amino acid having an insulin sensitivity enhancing activity. In particular, there is provided a preventive and/or therapeutic composition for liver diseases, characterized by containing at least one amino acid selected from the group consisting of aspartic acid, alanine, cystine, glutamine, glycine, histidine and salts thereof.

Description

肝疾患予防 ·治療用組成物  Liver disease prevention and treatment composition
技術分野  Technical field
[0001] 本発明は、肝疾患の予防及び/又は治療用組成物に関する。本発明は、またインス リン抵抗性関連肝疾患予防 Z又は治療用組成物に関する。  [0001] The present invention relates to a composition for preventing and / or treating liver diseases. The present invention also relates to a composition for preventing or treating insulin-related liver disease Z or treatment.
[0002] (発明の背景）  [0002] (Background of the Invention)
生活習慣病の増加は 21世紀の世界的な問題であるが、特に近年、糖尿病または 耐糖能異常、高血圧、脂質代謝異常 (高脂血症)等を伴った患者が増加している。こ れらは代謝症候群 (メタボリックシンドローム）、シンドローム X、インスリン抵抗性症候 群、マルチプルリスクファクター症候群などと呼ばれ、多くは肥満、特に内蔵型肥満と インスリン抵抗性に伴う高インスリン血症が特徴的で、動脈硬化、心血管イベントなど 重篤な病態への進展 ·発症リスクが高！ヽとされて!/、る。  The increase in lifestyle-related diseases is a global problem in the 21st century. In recent years, however, the number of patients with diabetes or impaired glucose tolerance, hypertension, lipid metabolism abnormality (hyperlipidemia), etc. has increased. These are called metabolic syndrome (metabolic syndrome), syndrome X, insulin resistance syndrome group, multiple risk factor syndrome, etc., and many are characterized by obesity, especially built-in obesity and hyperinsulinemia associated with insulin resistance And progress to serious pathological conditions such as arteriosclerosis and cardiovascular events · High risk of onset! It's ridiculous! /
またインスリン抵抗性は循環器イベントのみならず脂肪肝の発症や肝炎の進展予 後といった肝疾患にも関与していることが示唆されている。特に近年、アルコールを 摂取しないにも関わらず脂肪肝や肝機能異常を示す非アルコール性脂肪肝 (以下 N AFLD)が増加している力 これも代謝症候群関連疾患の一つのフヱノタイプと考えら れる。 NAFLDの 5〜20%は炎症性壊死、線維化といった肝組織所見を示す非アルコ ール性脂肪性肝炎（以下 NASH)に進行すると言われているが、 NASH患者は肝硬変 や肝癌などの重篤な肝疾患に至るリスクが高いことが疫学的に明ら力となっており、 N AFLDZNASHの予防、治療法の開発は医療上の重要な課題となっている（非特許 文献 1)。  It has also been suggested that insulin resistance is involved not only in cardiovascular events but also in liver diseases such as the development of fatty liver and the prognosis of hepatitis. In particular, the power of increasing fatty liver and nonalcoholic fatty liver (hereinafter referred to as N AFLD), which shows abnormal liver function despite not taking alcohol, is also considered to be one type of metabolic syndrome-related disease. It is said that 5 to 20% of NAFLD progresses to non-alcoholic steatohepatitis (NASH), which shows liver tissue findings such as inflammatory necrosis and fibrosis, but NASH patients have severe liver cirrhosis and liver cancer. Epidemiological evidence is that there is a high risk of developing liver disease, and the development of N AFLDZNASH prevention and treatment methods is an important medical issue (Non-patent Document 1).
NAFLD/NASHの病態ではインスリン抵抗性が悪ィ匕すると考えられており（非特許文 献 6)、インスリン受容体を肝特異的に破壊したマウスはインスリン抵抗性、耐糖能異 常、高インスリン血症を呈するとともに、肝機能異常が認められることが報告されてい る（非特許文献 7)。またインスリン抵抗性は TRB3、 LAR、グルコース一 6—フォスファ ターゼ (G6Pase)遺伝子と関連するとする以下の報告がある。すなわち、肥満、肝イン スリン抵抗性を示す db/dbマウスにおいてインスリンシグナル下流の AKTのリン酸ィ匕を 抑制する TRB3と呼ばれる遺伝子産物の発現が増加していること（非特許文献 8)、肥 満で肝インスリン抵抗性を示す Zuckerラットではインスリンシグナルを抑制するプロテ インチ口シンホスファターゼの一つである LARの発現が肝で増加して!/、ること（非特許 文献 9)が報告されている。また Zuckerラットや db/dbマウスで G6Paseの発現または活 性が亢進していることは多数報告されているが (非特許文献 10,11)、高脂肪食を与え たィヌで肝脂肪蓄積と同時に肝インスリン抵抗性が見られ、 G6Paseの発現が亢進し ていることも最近報告された (非特許文献 12)。従って、 LAR、 TRB3あるいは G6Pase のような遺伝子の肝細胞での発現を in vitroあるいは in vivoで直接あるいは間接的 に抑制するような組成物は、肝インスリン感受性を高めることでインスリン抵抗性を改 善し、 NAFLD/NASHといった肝疾患の予防.治療に有用である可能性がある。 Insulin resistance is considered to be poor in the pathology of NAFLD / NASH (Non-patent Document 6), and mice that have specifically destroyed the insulin receptor in the liver are insulin resistant, impaired glucose tolerance, and hyperinsulinemia. It has been reported that liver function abnormalities are observed (Non-patent Document 7). In addition, the following reports indicate that insulin resistance is related to the TRB3, LAR, and glucose-6-phosphatase (G6Pase) genes. That is, the expression of a gene product called TRB3 that suppresses AKT phosphate downstream of the insulin signal is increased in obese and hepatic insulin resistant db / db mice (Non-patent Document 8), It has been reported that in Zucker rats, which exhibit hepatic insulin resistance at the fullest level, the expression of LAR, one of the protein-synthetic phosphatases that suppress insulin signals, is increased in the liver! Yes. There are many reports that the expression or activity of G6Pase is enhanced in Zucker rats and db / db mice (Non-patent Documents 10 and 11). At the same time, hepatic insulin resistance was observed, and it was recently reported that G6Pase expression was increased (Non-patent Document 12). Therefore, a composition that directly or indirectly suppresses expression of genes such as LAR, TRB3, or G6Pase in hepatocytes in vitro or in vivo improves insulin resistance by increasing liver insulin sensitivity. However, it may be useful for the prevention and treatment of liver diseases such as NAFLD / NASH.
[0003] 現在インスリン抵抗性を改善する薬剤としてチアゾリジン系化合物（ピオグリタゾン、 ロジグリタゾン）が 2型糖尿病に対する治療薬として承認されており、これらはメタボリ ックシンドロームの諸症状にも有効であると 、う報告がある。さらにこれらが NAFLD/N ASHのようなインスリン抵抗性に関連する肝疾患へも有効である可能性を示唆する偵 察臨床試験結果も報告されている（非特許文献 2、 3)。しかし、これら薬剤の主たる作 用メカニズムは ppAR yの活性ィ匕による脂肪細胞への作用であり、これに起因する副 作用として体重増加が投与期間中のみならず投与終了後も続き肝障害が再燃する ことが問題とされている (非特許文献 2)。さらに、改善効果が見られるとした報告でも 組織学的に門脈域の炎症は増カロしているという報告 (非特許文献 3)や、肝での PPA 活性ィ匕はむしろ脂肪肝を惹起するという報告 (非特許文献 4、 5)からも、チアゾリ ジン系化合物の肝疾患を有する患者への長期投与の安全性は充分に確認されてい ない。すなわち、 NAFLD/NASHのようなインスリン抵抗性に関連する肝疾患に対し長 期的な予防効果や治療効果の高!、薬剤や安全な治療法は未だ確立されて!、な 、。 ところでアミノ酸は古くから知られている安全性の高い化合物であり、これまでにも アミノ酸を含む輸液については多くの栄養学的な報告がなされているが、代謝症候 群に関連する NAFLD/NASHのような肝疾患に関連する薬理学的な効果の報告は少 ない。 [0003] Thiazolidine-based compounds (pioglitazone, rosiglitazone) are currently approved as therapeutic agents for type 2 diabetes as drugs that improve insulin resistance, and these are effective for various metabolic syndrome symptoms. There is a report. In addition, the results of reconnaissance clinical trials suggesting that these may be effective for liver diseases related to insulin resistance such as NAFLD / NASH have also been reported (Non-patent Documents 2 and 3). However, the main mechanism of action of these drugs is the effect of ppAR y activity on adipocytes, and as a side effect resulting from this, weight gain continues not only during the administration period but also after the end of administration, and liver damage relapses. It is considered a problem (Non-Patent Document 2). Furthermore, even in reports that an improvement effect is seen, reports of histologically increased inflammation in the portal vein area (Non-patent Document 3), and PPA activity in the liver rather causes fatty liver. From these reports (Non-Patent Documents 4 and 5), the safety of long-term administration of thiazolidine compounds to patients with liver diseases has not been fully confirmed. In other words, long-term preventive and therapeutic effects on liver diseases related to insulin resistance such as NAFLD / NASH are high! Drugs and safe treatment methods are still established! By the way, amino acids are highly safe compounds that have been known for a long time, and there have been many nutritional reports on infusions containing amino acids, but NAFLD / NASH related to metabolic syndrome There are few reports of pharmacological effects related to such liver diseases.
[0004] 例えばアミノ酸の一つであるァスパラギン酸 (Asp)については以下のような報告が ある。特許文献 1では生活習慣病に対する metabolic uncoupling therapy(MUT)のた めの組成物が出願されており、例示されている 33種の構成成分の一つとしてァスパラ ギン酸が開示されている。また特許文献 2ではァスパラギン酸を含むアミノ酸と βダル カンに富む生薬由来の複数成分を含有する抗腫瘍活性、抗高脂血症活性、血圧降 下活性、血糖値降下活性を示す生理活性組成物が出願、開示されている。さらに特 許文献 3では 3-グァ -ジノプロピオン酸のシナプトゾームへの取込に対する拮抗作用 を示すィ匕合物が血糖降下作用を示す可能性があることから、糖尿病、肥満に有効性 を示す可能性のある 119ないし 128種の化合物が開示されている力 DL-Aspもこの表 に記載されている。しかしこれらの特許文献は、用途として肝疾患に対する効果を示 すものはなぐさらに L-Aspが単独で肝インスリン抵抗性を改善することを示す報告は ない。 [0004] For example, aspartic acid (Asp), one of the amino acids, has been reported as follows. In Patent Literature 1, metabolic uncoupling therapy (MUT) for lifestyle-related diseases A composition has been filed and aspartic acid is disclosed as one of the 33 exemplified components. In Patent Document 2, a physiologically active composition having an antitumor activity, an antihyperlipidemic activity, a blood pressure lowering activity, and a blood glucose lowering activity, comprising an amino acid containing aspartic acid and a plurality of components derived from a crude drug rich in β-dalcan. Have been filed and disclosed. Furthermore, in Patent Document 3, a compound that exhibits an antagonistic action on the incorporation of 3-gua-dinopropionic acid into synaptosomes may have a hypoglycemic action, and thus may be effective in diabetes and obesity. The force DL-Asp, in which 119 to 128 potential compounds are disclosed, is also listed in this table. However, these patent documents have no reports showing that L-Asp alone improves hepatic insulin resistance alone, in addition to those that show an effect on liver diseases.
また非特許文献 13-15にはァスパラギン酸と他のアミノ酸との塩ィ匕合物に関して、肝 疾患治療にっ ヽての記載があるが、 V、ずれもインスリン抵抗性に関連する肝疾患へ の効果に関する記載はな 、。また非特許文献 16は標識脂肪酸の短期的な肝への集 積に関する検討であるが、ァスパラギン酸力 Sインスリン抵抗性に関連する肝疾患の予 防 ·治療効果を示すか否かにっ 、ては示されて!/、な 、。  Non-patent documents 13-15 describe the treatment of liver diseases with regard to the salt compound of aspartic acid and other amino acids, but V, the gap is also related to the liver disease related to insulin resistance. There is no description about the effect of. Non-patent document 16 is a study on the short-term accumulation of labeled fatty acids in the liver. Whether or not it exhibits a preventive / therapeutic effect on liver disease related to aspartic acid S insulin resistance. Is shown! /
特許文献 1： US2004/0043013 A1 Patent Document 1: US2004 / 0043013 A1
特許文献 2：特開 2003-212775 Patent Document 2: JP 2003-212775
特許文献 3：特表平 6— 510760 Patent Document 3: Japanese Translation of Heisei 6-510760
非特許文献 1 : Gastroenterology, 2002; 123: 1702-1704、 1705-1725 Non-Patent Document 1: Gastroenterology, 2002; 123: 1702-1704, 1705-1725
非特許文献 2 : Hepatology, 2004; 39: 188-196 Non-Patent Document 2: Hepatology, 2004; 39: 188-196
非特許文献 3 : Hepatology, 2003; 38: 1008-1017 Non-Patent Document 3: Hepatology, 2003; 38: 1008-1017
非特許文献 4 :J. Lipid Res., 2002; 43: 1809-1817 Non-Patent Document 4: J. Lipid Res., 2002; 43: 1809-1817
非特許文献 5 : J. Clin. Invest., 2000; 106: 1221-1228 Non-Patent Document 5: J. Clin. Invest., 2000; 106: 1221-1228
非特許文献 6 : Semin. Liver Dis., 2004; 24: 3-20 Non-Patent Document 6: Semin. Liver Dis., 2004; 24: 3-20
非特許文献 7 : Mol Cell., 2000; 6: 87-97 Non-Patent Document 7: Mol Cell., 2000; 6: 87-97
非特許文献 8 : Science, 2003； 300: 1574-1577 Non Patent Literature 8: Science, 2003; 300: 1574-1577
非特許文献 9 : Diabetes, 2000; 49: 810-819. Non-Patent Document 9: Diabetes, 2000; 49: 810-819.
非特許文献 10 : J. Biol. Chem., 1998; 273: 31615—31620 非特許文献 11 : Diabetes, 1999; 48: 1579-1585 Non-Patent Document 10: J. Biol. Chem., 1998; 273: 31615-31620 Non-Patent Document 11: Diabetes, 1999; 48: 1579-1585
非特許文献 12 : Am J Physiol Endocrinol Metab., 2004 Nov 2 [Epub ahead of print] 非特許文献 13 : Fortschritte der Medizin (GERMANY), 1996; 114: 141-146, 非特許文献 14 : Zeitschrift for Allgemeinmedizin (GERMANY, WEST), 1973; 49: 469 Non-Patent Document 12: Am J Physiol Endocrinol Metab., 2004 Nov 2 [Epub ahead of print] Non-Patent Document 13: Fortschritte der Medizin (GERMANY), 1996; 114: 141-146, Non-Patent Document 14: Zeitschrift for Allgemeinmedizin ( GERMANY, WEST), 1973; 49: 469
-472 -472
非特許文献 15 : Recherche europeenne en toxicologie (FRANCE), 1978; 1: 303-309 非特許文献 16 : Therapie (FRANCE), 1966; 21: 719-731  Non-Patent Document 15: Recherche europeenne en toxicologie (FRANCE), 1978; 1: 303-309 Non-Patent Document 16: Therapie (FRANCE), 1966; 21: 719-731
発明の開示  Disclosure of the invention
[0006] 本発明は、肝疾患の予防や治療に有効な組成物を提供することを目的とする。  [0006] An object of the present invention is to provide a composition effective for the prevention and treatment of liver diseases.
より詳細に言えば、 TRB3のような肝インスリン抵抗性に関与する遺伝子の発現レべ ルを低下させるアミノ酸を発見し、これを含む NAFLD/NASHのようなインスリン抵抗性 に関連する肝疾患の予防及び Zまたは治療用組成物やキットを提供することを目的 とする。  More specifically, the discovery of amino acids that reduce the expression level of genes involved in hepatic insulin resistance such as TRB3, and the prevention of liver diseases related to insulin resistance such as NAFLD / NASH. And Z or to provide therapeutic compositions or kits.
本発明は、又、上記肝疾患予防及び Zまたは治療用組成物を含有する食品を提 供することを目的とする。  Another object of the present invention is to provide a food containing the composition for preventing and / or treating liver diseases.
本発明は、又、インスリン抵抗性関連疾患予防及び Zまたは治療用組成物、あるい はこれらを含有する機能性食品を提供することを目的とする。  Another object of the present invention is to provide a composition for preventing and / or treating insulin resistance related diseases, or a functional food containing them.
本発明は、又、インスリン感受性増強剤及びインスリン抵抗性改善剤を提供すること を目的とする。  Another object of the present invention is to provide an insulin sensitivity enhancer and an insulin resistance improver.
[0007] 本発明は、上記課題解決に向けて鋭意研究を重ねた結果、ある種のアミノ酸が、ィ ンスリン感受性増強作用を有し、又肝 TRB3または LARの少なくとも 1つの発現抑制作 用をも有し、このようなアミノ酸が、肝疾患予防及び治療に有効であるとの知見に基 づ 、てなされたものである。  [0007] As a result of intensive research aimed at solving the above problems, the present invention has a certain amino acid has an effect of enhancing insulin sensitivity, and also has an action of suppressing the expression of at least one of liver TRB3 or LAR. It has been made based on the knowledge that such amino acids are effective in the prevention and treatment of liver diseases.
すなわち、本発明は、インスリン感受性増強作用を有するアミノ酸を有効成分とする 肝疾患予防及び Zまたは治療用組成物を提供する。  That is, the present invention provides a composition for the prevention and Z or treatment of liver diseases comprising an amino acid having an insulin sensitivity enhancing action as an active ingredient.
本発明は、又、ァスパラギン酸、ァラニン、シスチン、グルタミン、グリシン、ヒスチジ ンおよびそれらの塩力 なる群力 選ばれる少なくとも 1つのアミノ酸を含有することを 特徴とする肝疾患予防及び Zまたは治療用組成物を提供する。 本発明は、又、 L-ァスパラギン酸および Zまたはその塩を単独の有効成分とするこ とを特徴とする肝疾患予防及び Zまたは治療用組成物を提供する。 The present invention also comprises at least one amino acid selected from aspartic acid, alanine, cystine, glutamine, glycine, histidine, and their salt strength, and a composition for preventing or treating liver disease, which is characterized in that Offer things. The present invention also provides a composition for preventing and treating liver disease or treatment comprising L-aspartic acid and Z or a salt thereof as a single active ingredient.
本発明は、又、 L-ァスパラギン酸および Zまたはその塩を有効成分として、第二の 有効成分として 1)ァラニン、シスチン、グルタミン、グリシン、ヒスチジンおよびそれら の塩、あるいは 2)アミノ酸及びその塩以外の肝疾患予防 ·治療薬の少なくとも一つを 含有するような肝疾患予防及び Zまたは治療用組成物、あるいはこれらを組み合わ せた肝疾患予防及び Zまたは治療用キットを提供する。  The present invention also includes L-aspartic acid and Z or a salt thereof as an active ingredient, and as a second active ingredient 1) alanine, cystine, glutamine, glycine, histidine and a salt thereof, or 2) other than an amino acid and a salt thereof A liver disease prevention and Z or treatment composition containing at least one of the following liver disease prevention / treatment drugs, or a combination of these liver disease prevention and Z or treatment kits is provided.
本発明は、又、上記肝疾患予防及び Zまたは治療用組成物を含有する食品を提 供する。  The present invention also provides a food containing the composition for preventing and treating liver disease or Z.
本発明は、又、インスリン感受性増強作用を有するアミノ酸を有効成分とするインス リン抵抗性関連疾患予防及び Zまたは治療用組成物、あるいはこれらを含有する食 品を提供する。  The present invention also provides an insulin resistance-related disease prevention and Z or treatment composition comprising an amino acid having an insulin sensitivity enhancing action as an active ingredient, or a food containing the same.
本発明は、又、ァスパラギン酸、ァラニン、シスチン、グルタミン、グリシン、ヒスチジ ンおよびそれらの塩力 なる群力 選ばれる少なくとも 1つを含むインスリン感受性増 強剤又はインスリン抵抗性改善剤を提供する。  The present invention also provides an insulin sensitivity enhancer or an insulin resistance improver comprising at least one selected from aspartic acid, alanine, cystine, glutamine, glycine, histidine, and the group power of their salt strength.
発明を実施するための最良の形態 BEST MODE FOR CARRYING OUT THE INVENTION
本発明における「インスリン作用」とはインスリンが体の組織にて代謝調節能を発揮 することをいう。より詳細に言えばインスリンが肝臓、筋肉、脂肪組織などのインスリン 感受性のある細胞のインスリン受容体に結合し、それらの細胞でグルコースの細胞内 への取り込み、エネルギー利用や貯蔵の促進、蛋白質合成促進、細胞増殖などの 細胞生物学的な働きを現すこと全般をさす。例えば in vitroでの G6Pase発現抑制によ る肝糖新生抑制やこれらを介した in vivoでの生理学的な血糖値の低下はインスリン 作用である。  “Insulin action” in the present invention means that insulin exerts metabolic regulation ability in body tissues. More specifically, insulin binds to insulin receptors of insulin-sensitive cells such as liver, muscle, and adipose tissue, and these cells take up glucose into cells, promote energy utilization and storage, and promote protein synthesis. In general, it refers to cell biology such as cell proliferation. For example, the suppression of hepatic gluconeogenesis by suppressing G6Pase expression in vitro and the decrease in physiological blood glucose level in vivo via these are insulin actions.
本発明における「インスリン抵抗性」とは「インスリン感受性の低下」を ヽ、より詳細 かつ細胞生物学的に言えば、インスリン受容体数の減少、インスリン拮抗物質の存在 、その他インスリン受容体を介する細胞内への情報伝達能力の低下状態をいう。例 えばインスリンシグナル伝達の第一段階である基質リン酸ィ匕を脱リン酸ィ匕する LARや 同じくインスリンシグナル伝達過程の一部作用に必須な AKTリン酸ィ匕を抑制する TRB 3の発現の増加は、細胞レベルでのインスリン抵抗性の存在 (インスリン感受性の低 下)を意味する。 In the present invention, “insulin resistance” refers to “decrease in insulin sensitivity”. More specifically and cell biologically speaking, a decrease in the number of insulin receptors, presence of an insulin antagonist, other cells mediated by insulin receptors A state of reduced information transmission ability. For example, LAR, which dephosphorylates the substrate phosphate, which is the first step in insulin signaling, and TRB, which also inhibits AKT phosphate, which is essential for a part of the insulin signaling process An increase in expression of 3 implies the presence of insulin resistance at the cellular level (decreased insulin sensitivity).
また本発明における「インスリン抵抗性」、「インスリン感受性の低下」は、生理学的 (i n vivo)には血中のインスリン濃度に見合ったインスリン作用（血糖値の低下）が得ら れな ヽ状態をさす。臨床的には HOMA-IR (空腹時血中インスリン値と空腹時血糖値 の積に比例する）や QUICKIが簡便な指標として用いられる。また糖負荷試験、インス リン負荷試験、グルコースクランプ法、ミニマルモデル法などを単独および組み合わ せることでより詳細、厳密に評価することもできる。例えば病態動物においても空腹時 血中インスリン値と空腹時血糖値の積の上昇、糖負荷時の高インスリン分泌 Z耐糖 能低下はインスリン抵抗性の存在 (インスリン感受性の低下)を意味する。  In addition, “insulin resistance” and “decrease in insulin sensitivity” in the present invention are physiological (in vivo) conditions in which an insulin action (decrease in blood glucose level) corresponding to the insulin concentration in blood cannot be obtained. Sure. Clinically, HOMA-IR (which is proportional to the product of fasting blood insulin level and fasting blood glucose level) and QUICKI are used as simple indicators. In addition, a more detailed and rigorous evaluation can be performed by combining a glucose tolerance test, an insulin tolerance test, a glucose clamp method, a minimal model method, and the like. For example, even in pathological animals, an increase in the product of fasting blood insulin level and fasting blood glucose level, and high insulin secretion during glucose load, decreased Z glucose tolerance means the presence of insulin resistance (decrease in insulin sensitivity).
[0009] 本発明における「インスリン感受性増強作用」とは上記インスリン感受性を増強する 作用であればどのような機作に基づくものでも良ぐ直接もしくは間接に細胞もしくは 生体のインスリン抵抗性を改善しインスリンの作用を高めるものであれば良い。のみ ならず、インスリン作用のすべてあるいは一部をインスリン非存在下で代替する作用、 および Zまたはインスリン供給を補う作用をあわせ持っていても良ぐ最終的に in vivo でのインスリン作用不足を改善する作用であれば良い。すなわち「インスリン感受性 増強作用」は、「インスリン抵抗性」を改善する効果も有する。  [0009] In the present invention, the "insulin sensitivity enhancing action" may be based on any mechanism as long as it is an action that enhances insulin sensitivity. Anything that enhances the function of the above is acceptable. It may be combined with the action of substituting all or part of the insulin action in the absence of insulin, and supplementing Z or insulin supply, ultimately improving the insufficiency of insulin action in vivo It only has to be an action. That is, the “insulin sensitivity enhancing action” also has the effect of improving “insulin resistance”.
本発明における「代謝症候群 (メタボリック'シンドローム）」は狭義には米国コレステ ロール教育プログラム（National Cholesterol Education Program; NCEP)成人治療委 員会（Adult Treatment Panel; ATP)が 2001年に発表した下記表 Aの診断基準ガイド ライン (NCEP-ΑΤΡΙΠ)を満たす疾患、患者を指す。  In the present invention, “metabolic syndrome” in the narrow sense is the following Table A, published in 2001 by the National Cholesterol Education Program (NCEP) Adult Treatment Panel (ATP). Refers to patients with diseases that meet the diagnostic criteria guidelines (NCEP-IV).
[0010] A [0010] A
Figure imgf000007_0001
Figure imgf000007_0001
1)腹部肥満 (ウェスト周囲径） 男性 > 102cm  1) Abdominal obesity (waist circumference) Male> 102cm
女性 >88cm  Female> 88cm
2)トリグリセリド ≥150mg/dL  2) Triglyceride ≥150mg / dL
3) HDLコレステロール 男性 <40mg/dL  3) HDL cholesterol Male <40mg / dL
女性 < 50mg/dL 4)血圧 収縮期 ≥130mmHg Female <50mg / dL 4) Blood pressure systolic ≥130mmHg
拡張期 ≥85mmHg  Diastolic ≥85mmHg
5) 随 ίΐΠ.糖 ≥H0mg/dL 5) incidental ίΐΠ. Sugar ≥H0m g / dL
上記危険因子 5項目中 3項目以上に該当する場合、代謝症候群と診断する (JAMA.2 001: 285: 2486-2497) Diagnosis of metabolic syndrome when 3 or more of the above 5 risk factors apply (JAMA.2 001: 285: 2486-2497)
また本発明における「代謝症候群関連疾患」とはシンドローム X、インスリン抵抗性 症候群、マルチプルリスクファクター症候群などの統合的概念としての WHO提唱に 準じ、上記 ΑΤΡΠΙの危険因子、基準値に限定されず、肥満（内臓脂肪蓄積)、インスリ ン抵抗性 (高インスリン血症)とこれに関連する 2型糖尿病 (耐糖能異常)、脂質代謝 異常（高トリグリセリド血症、 VLDL-トリグリセリド上昇、 HDL-コレステロール低下）、脂 肪肝、高血圧、尿中微量アルブミンなど、共通する分子病態基盤に立脚すると考えら れる疾患 ·症状を包括した呼称として用いる。  In addition, the “metabolic syndrome-related disease” in the present invention is not limited to the above-mentioned risk factors and standard values of wrinkles according to the WHO proposal as an integrated concept such as syndrome X, insulin resistance syndrome, multiple risk factor syndrome, etc. (Visceral fat accumulation), insulin resistance (hyperinsulinemia) and related type 2 diabetes (abnormal glucose tolerance), abnormal lipid metabolism (hypertriglyceridemia, VLDL-triglyceride elevation, HDL-cholesterol decline), It is used as a comprehensive name for diseases and symptoms that are thought to be based on common molecular pathologies such as fatty liver, hypertension, and urinary microalbumin.
本発明における「非アルコール性脂肪肝 (NAFLD) Z非アルコール性脂肪性肝炎（ NASH)」とは、例えば米国消化器病学会及び米国肝臓病学会が 2002年に発表した ステートメント並びにテク-カルレビュー（非特許文献 1)で提示したように、脂肪肝、 炎症、線維化で診断 (Grading. Staging)され、肥満、糖尿病、高脂血症、インスリン抵 抗性と関連する疾患とする。  In the present invention, “non-alcoholic fatty liver (NAFLD) Z non-alcoholic steatohepatitis (NASH)” refers to, for example, a statement and technical review published by the American Society of Gastroenterology and the American Society of Liver Disease in 2002. As presented in Non-Patent Document 1), it is diagnosed with fatty liver, inflammation, and fibrosis (Grading. Staging), and it is a disease associated with obesity, diabetes, hyperlipidemia, and insulin resistance.
本発明における「インスリン抵抗性関連肝疾患」とは、その発症または病態の形成 にお 、てインスリン抵抗性が主に関与して ヽる肝疾患を! ヽ、例えば非アルコール 性脂肪肝 (NAFLD)や非アルコール性脂肪性肝炎 (NASH)などが含まれる。  The term “insulin resistance-related liver disease” in the present invention refers to a liver disease in which insulin resistance is mainly involved in its onset or pathogenesis! ヽ For example, non-alcoholic fatty liver (NAFLD) And nonalcoholic steatohepatitis (NASH).
本発明における「インスリン抵抗性関連疾患」とは、その発症または病態の形成に お 、てインスリン抵抗性が主に関与して 、る肝疾患を 、 、、例えばメタボリックシンド ロームや代謝症候群関連疾患などを含む。  The “insulin resistance-related disease” in the present invention is a liver disease mainly related to insulin resistance in its onset or pathogenesis, such as metabolic syndrome and metabolic syndrome-related diseases. including.
本発明における「疾患予防及び Zまたは治療」とは上記疾患と診断されな 、こと、 上記疾患と診断された者がその定義を外れること、上記疾患と診断された測定値の 異常が改善されること、もしくは上記疾患と診断された異常の増悪'進展を遅らせるこ とをさし、例えば空腹時血糖値、空腹時インスリン値、耐糖能、肝脂肪量の維持'改 善をいう。 [0012] インスリン感受性増強作用を有するアミノ酸は、細胞、例えばラット肝癌由来細胞株 Faoを、 1種類もしくは数種類のアミノ酸を添加した培地で 30分から 1週間、より望ましく は 3時間培養し、そのインスリン感受性を、例えば TRB3の発現レベルを指標に、ァミノ 酸を含まない培地で培養した細胞と比較測定することで見出すことができる。 Fao細 胞は ί列 ば European し ollection of Animal し ellし ultures (Porton Down, UK); ^り Eし ACC No. 89042701として入手することができる。 In the present invention, “disease prevention and Z or treatment” means that the above-mentioned disease is not diagnosed, that the person diagnosed with the above-mentioned disease deviates from the definition, and abnormality in the measured value diagnosed with the above-mentioned disease is improved. Or, exacerbation of the abnormality diagnosed as the above-mentioned disease means delaying the progress, for example, maintaining or improving fasting blood glucose level, fasting insulin level, glucose tolerance, liver fat mass. [0012] An amino acid having an insulin-sensitizing effect is obtained by culturing cells, for example, rat liver cancer-derived cell line Fao in a medium supplemented with one or several amino acids for 30 minutes to 1 week, more preferably 3 hours. Can be found, for example, by comparative measurement with cells cultured in a medium not containing amino acid, using the expression level of TRB3 as an index. Fao cells can be obtained as ACC No. 89042701 in European and ollection of Animals and ultures (Porton Down, UK);
あるいはあるアミノ酸が実際にインスリン作用を増強するか否かを細胞、例えばラッ ト肝癌由来細胞株 Faoを、 1種類もしくは数種類のアミノ酸を添加した培地で 30分から 1週間、より望ましくは 6時間培養し、そのインスリン様作用ならびにインスリン作用増 強作用を、例えば G6Paseの発現レベルの抑制を指標に、アミノ酸を含まない培地で 培養した細胞と比較測定することで確認することができる。  Alternatively, to determine whether an amino acid actually enhances insulin action, cells such as rat liver cancer cell line Fao are cultured in a medium supplemented with one or several amino acids for 30 minutes to 1 week, more preferably 6 hours. The insulin-like action and the insulin action-enhancing action can be confirmed by, for example, comparative measurement with cells cultured in a medium containing no amino acid, with suppression of the expression level of G6Pase as an index.
あるいは特定のアミノ酸が実際にインスリン作用を増強する力否かを、インスリン作 用が不足している患者あるいは病態動物、例えば GKラットに対し、特定のアミノ酸を 1 日から 1年、望ましくは 3〜8週間投与し、インスリン作用を例えば空腹時血糖、空腹時 インスリンの低下、経口糖負荷試験による耐糖能の改善、及び肝臓における TRB3、 L AR、 G6Pase遺伝子の発現低下などを指標に、アミノ酸を投与しない群と比較測定す ることで確認することができる。  Alternatively, whether or not a specific amino acid actually enhances insulin action can be determined by applying a specific amino acid from 1 day to 1 year, preferably from 3 to 1 for patients or pathological animals lacking insulin action, such as GK rats. Administered for 8 weeks and administered amino acids with indicators of insulin action such as fasting blood glucose, decreased fasting insulin, improved glucose tolerance by oral glucose tolerance test, and decreased expression of TRB3, LAR, G6Pase genes in liver This can be confirmed by comparison with the group that does not.
[0013] 特定のアミノ酸が肝疾患、特に NAFLD/NASHを改善するかについては、脂肪肝の 増加に脂肪摂取量の増加が大きな要因になっているとされていることから、非アルコ ール性脂肪肝患者あるいは高脂肪食を与えて作成した病態動物に対し特定のァミノ 酸を 1日から 1年、望ましくは 3〜6週間投与し、肝トリグリセリド含量、及び肝臓におけ る脂肪合成関連酵素遺伝子 (例えばリンゴ酸酵素 (Malic Enzyme)、ステアロイル CoA デサチユラーゼ 1 (SCD1) )や脂肪燃焼系（ β酸化)制御関連転写因子、酵素遺伝子 (PPAR a、ァシル CoAォキシダーゼ（ACO)、カル-チンパルミトイルトランスフェラー ゼ 1 (CPT1) )、あるいは脂質代謝制御関連に関連する肝細胞特異的転写因子遺伝 子 (肝細胞核因子 4 a (HNF4 a ) )の発現などを指標に、アミノ酸を投与しない群と比 較測定することで確認することができる。 [0013] Regarding whether specific amino acids improve liver disease, especially NAFLD / NASH, it is considered that the increase in fat intake is a major factor in the increase in fatty liver. A specific amino acid is administered for 1 to 1 year, preferably 3 to 6 weeks, to a fatty liver patient or a diseased animal prepared by giving a high fat diet, and hepatic triglyceride content, and adipose-related enzyme gene in the liver (For example, malic enzyme (Malic Enzyme), stearoyl CoA desaturase 1 (SCD1)), fat burning system (β oxidation) regulation-related transcription factor, enzyme gene (PPAR a, acyl CoA oxidase (ACO), cal-tin palmitoyltransferase) 1 (CPT1)), or the expression of the hepatocyte-specific transcription factor gene (hepatocyte nuclear factor 4a (HNF4a)) related to lipid metabolism control, etc. Can be confirmed.
特定のアミノ酸力 Sメタボリックシンドロームを改善するかにっ 、ては、メタボリックシン ドローム様の病態動物に対し、特定のアミノ酸を 1日から 1年、望ましくは 3〜8週間投 与し、絶食時血糖、トリグリセリド等メタボリックシンドロームの異常判定基準項目値を 指標に、アミノ酸を投与しない群と比較測定することで確認することができる。脂肪肝 およびインスリン感受性の低下したメタボリックシンドローム様の病態動物は、正常、 あるいは GKラットのような病態動物に、蔗糖、果糖、あるいは高脂肪食などを 1日から 1年、望ましくは 2〜8週間投与し負荷することで、作成することができる。 Specific amino acid strength S Metabolic syndrome is improved. A specific amino acid is administered to Drome-like pathological animals for 1 to 1 year, preferably 3 to 8 weeks, and no amino acid is administered using abnormal metabolic criteria items such as fasting blood glucose and triglycerides as indicators. This can be confirmed by comparative measurement with the group. Metabolic syndrome-like animals with reduced fatty liver and insulin sensitivity may be normal or diseased animals such as GK rats with sucrose, fructose, or high-fat diet for 1 to 1 year, preferably 2 to 8 weeks. Can be created by administration and loading.
また予防効果については、特定のアミノ酸を高脂肪食と同時に混餌もしくは経口投 与することで、治療効果同様確認することができる。  The preventive effect can be confirmed by administering a specific amino acid at the same time as a high fat diet or by oral administration, as well as the therapeutic effect.
本発明で用いるアミノ酸としては、このような効果を有するものであれば、天然に存 在する、しないに関わらず、またアミノ酸自体は無論のこと、その薬学的に許容される 塩類を使用できる。具体的には、例えば、ァスパラギン酸、ァラニン、シスチン、ダル タミン、グリシン、ヒスチジンおよびそれらの塩からなる群力も選ばれる 1種又は 2種以 上のアミノ酸があげられ、特にこれらのアミノ酸は、 L体であるのが好ましい。これらの うちでも、特に L—ァスパラギン酸が好ましい。また塩を用いる場合は、好ましくは L- ァスパラギン酸のアルカリ金属塩あるいはアルカリ土類金属塩、例えば Lーァスパラ ギン酸ナトリウム塩が好まし 、。  As long as it has such an effect, the amino acid used in the present invention may be any pharmaceutically acceptable salt, regardless of whether it exists in nature or not. Specifically, for example, one or more amino acids from which group power consisting of aspartic acid, alanine, cystine, dartamine, glycine, histidine and their salts are also selected are mentioned. The body is preferred. Of these, L-aspartic acid is particularly preferred. When a salt is used, an alkali metal salt or alkaline earth metal salt of L-aspartic acid, for example, sodium L-aspartate is preferred.
本発明の組成物は、アミノ酸として上記アミノ酸及び Z又はその塩のみとすることが できるが、上記以外のアミノ酸を含有させることができる。上記以外のアミノ酸を含有 させる場合には、その量はアミノ酸全体の 50重量％以下とするのが好ましい。  The composition of the present invention can contain only the above amino acids and Z or a salt thereof as amino acids, but can contain amino acids other than those described above. When an amino acid other than the above is contained, the amount is preferably 50% by weight or less of the whole amino acid.
さらに、本発明の組成物には、上記成分にカ卩えて、薬理学的に許容し得る各種の 製剤用物質を製剤学上許容される担体 (補助剤)として含むこともできる。  Furthermore, the composition of the present invention may contain various pharmacologically acceptable pharmaceutical substances as a pharmaceutically acceptable carrier (adjuvant) in addition to the above components.
本発明の組成物は、例えば、経口投与、腹腔内投与、経皮的投与、吸入投与等各 種の投与形態に調製することができる。具体的には、適当な固形又は液状の製剤形 態、例えば顆粒、粉剤、被覆錠剤、錠剤、（マイクロ)カプセル、坐剤、シロップ、ジュ ース、懸濁液、乳濁液、滴下剤、輸液、注射用溶液、活性物質の放出を延長する製 剤等を挙げることができる。  The composition of the present invention can be prepared in various dosage forms such as oral administration, intraperitoneal administration, transdermal administration, and inhalation administration. Specifically, suitable solid or liquid preparation forms such as granules, powders, coated tablets, tablets, (micro) capsules, suppositories, syrups, juices, suspensions, emulsions, drops, Examples include infusion solutions, injection solutions, and preparations that prolong the release of active substances.
本発明に使用するアミノ酸の摂取量については、症状に応じて適当に選択される 力 経口投与の場合は成人患者で 1日当たり、アミノ酸の正味重量で表して 1日当た り lg以上、好ましくは 3g〜300g程度、更に好ましくは 3〜20g程度摂取することがで きる。また、重篤な場合には更に増量することもできる。投与の回数、時期について は、数日に 1回でも、また 1日 1回でも可能である力 通常は 1日当たり数回、例えば 2 〜4回に分けて投与される。 The intake of amino acids used in the present invention is appropriately selected according to the symptom. In the case of oral administration, the daily dose is expressed in terms of the net weight of amino acids in an adult patient per day. More than 1 lg, preferably about 3 g to 300 g, more preferably about 3 to 20 g. In severe cases, the dose can be further increased. Regarding the frequency and timing of administration, it can be applied once every few days or once a day. Usually, it is administered several times a day, for example, 2-4 times.
本発明では、上記アミノ酸を単独で、又は他の薬剤と組み合わせた形態で使用す ることができる。組み合わせる薬剤としては他のアミノ酸や肝疾患予防 ·治療薬があげ られる。ァスパラギン酸と組み合わせる場合、他のアミノ酸としてはァラニン、シスチン 、グルタミン、グリシン、ヒスチジンおよびそれらの塩などが好ましいものとしてあげられ 、特にァラニンが好ましい。又、アミノ酸及びその塩以外の肝疾患予防 ·治療薬として は、ウルソデォキシコール酸、グリチルリチン、ベタインといった肝庇護剤（Hepatoprot ectant)の他、肝疾患の予防'治療効果が報告されているピオグリタゾン、ロジグリタゾ ン、メトホルミンなどの糖尿病治療薬、クロフイブレート、ゲムフイブ口ジル、アト口バス タチン、口サルタン、プロブコール、ィコサペント酸ェチル、ホスファチジルコリン、ポリ ェンホスファチジルコリンなどの循環器疾患治療薬、シブトラミン、オルリスタツトなど の抗肥満薬、ビタミン C、ビタミン Eなどの抗酸化剤、ペントキシフィリン、抗 TNF抗体 などの抗炎症剤などが好ましいものとしてあげられる。  In the present invention, the above amino acids can be used alone or in combination with other drugs. Examples of drugs to be combined include other amino acids and liver disease prevention / treatment drugs. When combined with aspartic acid, preferred examples of other amino acids include alanine, cystine, glutamine, glycine, histidine, and salts thereof, and alanine is particularly preferred. In addition to amino acids and their salts, other preventive and therapeutic agents for liver disease have been reported, including hepatoprot ectant such as ursodeoxycholic acid, glycyrrhizin, and betaine, as well as preventive and therapeutic effects on liver disease. Antidiabetic drugs such as pioglitazone, rosiglitazone, metformin, clofibrate, gemfib mouth gill, atto mouth bastatin, mouth sultan, probucol, icosapentate ethyl, phosphatidylcholine, polyphosphatidylcholine and other circulatory disease drugs, sibutramine, Preferred are anti-obesity drugs such as orlistat, antioxidants such as vitamin C and vitamin E, and anti-inflammatory drugs such as pentoxifylline and anti-TNF antibody.
また上記アミノ酸を上記以外の代謝症候群やインスリン抵抗性に関連する疾患に 対する治療薬、例えば糖尿病に対する速効型インスリン分泌促進剤であるナテグリニ ドと組み合わせた形態で使用することもできる。  In addition, the above amino acids can be used in a form combined with a therapeutic agent for other diseases related to metabolic syndrome and insulin resistance, such as nateglinide, which is a rapid-acting insulin secretagogue for diabetes.
上記薬剤の組み合わせは、 1つの医薬組成物中に両者を共に含有する形態でも、 それぞれの成分を含有する 2つの医薬組成物を同時に又は多少の間隔をお 、て投 与する形態の 、ずれでもよ 、。他のアミノ酸と組み合わせる場合は生体内で分解し てこれらのアミノ酸になる前駆体、例えばペプチドを用いても良!、。  The combination of the above drugs may be in a form containing both of them in one pharmaceutical composition or in a form in which two pharmaceutical compositions containing each component are administered simultaneously or at some interval. Yo ... When combined with other amino acids, precursors such as peptides may be used, which decompose in vivo to become these amino acids.
併用の場合、組成物中の他の薬剤の量は、個々の薬剤の有効量として好適な使用 量や投与量を採用することができる。例えば L-ァスパラギン酸と L-ァラニンを組み合 わせる場合、 L-ァスパラギン酸は 20mg- 50gZ日、好ましくは 0.1- 40gZ日、より好まし くは 1回 3-10gを 1日 1-4回経口投与することが望ましい。一方 L-ァラニンは 5-50gZ 日、より好ましくは 1回 6gを 1日 1-3回経口投与することが望ま 、。 一方、本発明において食品、特に機能性食品とする場合、例えばサプリメントとして 顆粒、粉剤、被覆錠剤、錠剤、（マイクロ)カプセルとしても良ぐまた飲料、ゼリー、ビ スケットなどの菓子、プリン、ヨーグルトなどの食品、あるいはスラリー状、ペースト状、 乳液状の医療用流動食などに本発明の予防及び Zまたは治療用組成物を含有させ 、そのパッケージに、用途あるいは効能を表記したものがあげられる。 In the case of combined use, the amount of other drugs in the composition can be used in an appropriate amount or dosage as an effective amount of each drug. For example, when combining L-aspartic acid and L-alanin, L-aspartic acid is 20 mg-50 gZ day, preferably 0.1-40 gZ day, more preferably 3-10 g once a day 1-4 times a day. Oral administration is desirable. On the other hand, L-alanine should be administered orally at 5-50 gZ days, more preferably 6 g at a time, 1-3 times a day. On the other hand, when foods, particularly functional foods, are used in the present invention, for example, granules, powders, coated tablets, tablets, (micro) capsules may be used as supplements, and confectionery such as beverages, jellies, biscuits, puddings, yogurts, etc. In addition, the preventive and Z or therapeutic composition of the present invention may be contained in a food, a slurry, a paste, or a milky liquid liquid for medical use, and the use or efficacy described in the package.
ノ ッケージとしては、例えば一定量以上含有するアミノ酸及び zまたはその用途 · 効能や飲食方法などに関する説明事項を記載したいわゆる能書があげられる。 用途 '効能としては、代謝症候群、メタボリックシンドローム、インスリン抵抗性、肝疾 患、肝機能異常、脂肪肝、 NAFLD、 NASH,体重増加、肥満、腹部肥満、内臓型肥 満、内臓脂肪蓄積、糖尿病、耐糖能異常、高血圧、脂質代謝異常 (高脂血症)、高ィ ンスリン血症、動脈硬化、心血管イベントならびにこれらに関係する症状の予防ゃ改 善、あるいはこれらに関係する臨床指標や生化学検査値の悪ィ匕防止や改善があげ られる。  Examples of the knockout include a so-called Noh book that describes the amino acids contained in a certain amount or more and z or explanations regarding its use, efficacy, and eating and drinking method. Applications' Indications include metabolic syndrome, metabolic syndrome, insulin resistance, liver disease, liver dysfunction, fatty liver, NAFLD, NASH, weight gain, obesity, abdominal obesity, visceral fat accumulation, visceral fat accumulation, diabetes, Impaired glucose tolerance, hypertension, dyslipidemia (hyperlipidemia), hyperinsulinemia, arteriosclerosis, cardiovascular events and the prevention of related symptoms, or clinical indicators and biochemistry related to these This can prevent or improve the inspection value.
食品とする場合のアミノ酸の含量は食品全体に対し、 0.1-100重量％、好ましくは 3 重量％以上、より好ましくは 6〜： LOO重量％である。又、アミノ酸含量が 70〜： LOO重 量％である食品、すなわち主成分が本発明のアミノ酸のみである食品 (機能性食品、 サプリメントなど)も好ましい。又、アミノ酸の含量が 3〜6重量0 /0であるのも好ましい。 アミノ酸の含量は、食品の形態及び一日あたりの摂取量等により、本発明の効果を奏 するように任意に設定することも可能である。本発明では、これらのアミノ酸として本 発明で用いるアミノ酸のみを用いることができる力 これ以外のアミノ酸 (他のアミノ酸 という）を併用することができる。ここで、他のアミノ酸を併用する場合、アミノ酸全体の 50重量％以下とするのが好ましい。 The content of amino acids in the case of food is 0.1 to 100% by weight, preferably 3% by weight or more, more preferably 6 to LOO% by weight, based on the whole food. In addition, foods having an amino acid content of 70 to: LOO weight%, that is, foods (functional foods, supplements, etc.) whose main component is only the amino acid of the present invention are also preferred. Further, the content of amino acids is also preferred 3-6 wt 0/0. The content of amino acids can be arbitrarily set so as to achieve the effects of the present invention, depending on the form of food and the daily intake. In the present invention, the ability to use only the amino acids used in the present invention as these amino acids can be used in combination with other amino acids (referred to as other amino acids). Here, when other amino acids are used in combination, it is preferably 50% by weight or less of the total amino acids.
又、本発明で用いるアミノ酸としては、アミノ酸自体を用いるのが好ましいが、ァミノ 酸自体に限定されず、例えば前記の L-ァスパラギン酸には加水分解反応、特に生 体内加水分解反応により L-ァスパラギン酸になり得る物質、例えば L-ァスパラギン酸 を構成単位に持つ蛋白質やペプチドを用いてもよ ヽ。  The amino acid used in the present invention is preferably the amino acid itself, but is not limited to the amino acid itself. For example, the above-mentioned L-aspartic acid is subjected to hydrolysis reaction, particularly L-asparagine by in vivo hydrolysis reaction. A substance capable of becoming an acid, for example, a protein or peptide having L-aspartic acid as a structural unit may be used.
又、本発明で対象とする食品には、前記の用途'効能を単独で示さない乳化剤や 香料を含む機能性食品も含まれる。 本発明で対象とする食品として、前記の用途'効能を示し、他の有効成分を含まな Vヽ L-ァスパラギン酸またはその塩単独のサプリメントの場合は、例えば L-ァスパラギ ン酸単独の機能性食品の場合は、通常成人で 1日当たり lg以上、好ましくは 3g以上 、更に好ましくは 3〜40gを摂取することができるような商品形態の組成物が好ましい また L-ァスパラギン酸を含有させた医療用食品、保健機能食品、栄養機能食品、 特定保健用食品といった機能性食品の場合は一定量以上の含有量、例えば 1食で の摂取単位量あたり 2gを越える L-ァスパラギン酸を含み、 L-ァスパラギン酸以外の 蛋白質 ·アミノ酸が 1食あたり 10g以下であるような形態の経口用濃厚流動食があげら れるが、必ずしもこれに限定されるものではない。 In addition, the foods targeted by the present invention include functional foods containing emulsifiers and fragrances that do not exhibit the above-mentioned uses. In the case of V ヽ L-aspartic acid or a salt alone supplement that exhibits the above-mentioned effects and does not contain other active ingredients as a food subject to the present invention, for example, the functionality of L-aspartic acid alone In the case of food, a composition in the form of a product that can usually ingest lg or more, preferably 3 g or more, more preferably 3 to 40 g per day for an adult is preferable. Also, for medical use containing L-aspartic acid In the case of functional foods such as foods, functional health foods, functional nutritional foods, and foods for specified health use, L-asparagine contains more than a certain amount, for example, more than 2 g of L-aspartic acid per unit of intake per meal. Examples include, but are not necessarily limited to, concentrated liquid foods for oral use in which the amount of protein other than acids and amino acids is 10 g or less per meal.
[0016] 本発明によれば、インスリン感受性増強作用を有するアミノ酸を有効成分とする肝 疾患予防及び Zまたは治療用組成物、特に、非アルコール性脂肪肝もしくは非アル コール性脂肪性肝炎の予防及び治療に有効な組成物が提供される。又、ァスパラギ ン酸、ァラニン、シスチン、グルタミン、グリシン、ヒスチジンが肝インスリン抵抗性に関 与する TRB3の発現を抑制することができ、さらにその中の L-ァスパラギン酸が生体 内投与でも肝 TRB3の発現を抑制しインスリン抵抗性を改善し、病態動物における肝 脂肪蓄積を改善し、更に体重増加等の副作用がないとの利点がある。 [0016] According to the present invention, a liver disease prevention and Z or therapeutic composition comprising an amino acid having an insulin sensitivity enhancing action as an active ingredient, particularly prevention of nonalcoholic fatty liver or nonalcoholic steatohepatitis and A therapeutically effective composition is provided. Furthermore, aspartate, alanine, cystine, glutamine, glycine, and histidine can suppress the expression of TRB3, which is related to liver insulin resistance. It has the advantages of suppressing expression, improving insulin resistance, improving liver fat accumulation in diseased animals, and having no side effects such as weight gain.
次に実施例に基づいて本発明を説明する。  Next, this invention is demonstrated based on an Example.
実施例  Example
[0017] (実施例 1) (培養肝細胞に対するアミノ酸の TRB3発現抑制効果）  [Example 1] (Inhibitory effect of TRB3 expression of amino acids on cultured hepatocytes)
ラット肝癌由来細胞株 Faoの TRB3発現に対するアミノ酸の抑制効果を 22種類のアミ ノ酸について検討した。  The inhibitory effect of amino acids on the expression of TRB3 in rat liver cancer-derived cell line Fao was examined for 22 amino acids.
Fao細胞 2xl05cells/wellを 24well plateに播き、 10%牛胎児血清含有 RPMI1640培地 (ナカライテスタ株式会社 Code No.30264-85 )で 37°CCO 5%条件下 1晚培養後、翌 Fao cells 2xl0 5 cells / well are seeded on a 24-well plate, cultured in RPMI 1640 medium (Nacalai Tester Code No. 30264-85) containing 10% fetal calf serum, 1% culture at 37 ° CCO 5%, then the following
2  2
日に 10%牛胎児血清含有 RPMI1640培地（通常培地）またはアミノ酸を全て除いた 10 %牛胎児血清含有 RPMI培地 (マイナスアミノ酸培地）、またはこれに 1種類の L-ァミノ 酸 Xxx(濃度 ImM)を添カ卩した培地（マイナスアミノ酸 +L-Xxx)に交換し、 37°CCO 5  10% fetal bovine serum-containing RPMI1640 medium (normal medium) or 10% fetal bovine serum-containing RPMI medium (minus amino acid medium) excluding all amino acids, or one L-amino acid Xxx (concentration ImM) Replace with supplemented medium (minus amino acid + L-Xxx), 37 ° CCO 5
2 2
%条件下でさらに 3時間培養した。マイナスアミノ酸培地の組成は表 1に示した。また通 常培地はマイナスアミノ酸培地に表 2記載のアミノ酸を添加した培地である。 The cells were further cultured for 3 hours under% conditions. The composition of the minus amino acid medium is shown in Table 1. Again The normal medium is a medium obtained by adding the amino acids listed in Table 2 to a minus amino acid medium.
[0018] 表 1 [0018] Table 1
Inorganic salts g/liter  Inorganic salts g / liter
Calcium Nitrate■ 4H 0.10  Calcium Nitrate4H 0.10
Magnesium Sulfate 0.04884  Magnesium Sulfate 0.04884
Potassium Chloride 0.40  Potassium Chloride 0.40
Sodium Chloride 6.00  Sodium Chloride 6.00
Sodium Phosphate Dibasic 0.80  Sodium Phosphate Dibasic 0.80
Vitamins mg/liter  Vitamins mg / liter
D-Biotin 0.20  D-Biotin 0.20
Choli ne Chloride 3.00  Choli ne Chloride 3.00
Folic Acid 1.00  Folic Acid 1.00
myo— Inositol 35.00  myo— Inositol 35.00
Niacinamide 1.00  Niacinamide 1.00
D— Pantothenic Acid ■Ca 0.25  D— Pantothenic Acid ■ Ca 0.25
P-Amino Benzoic Acid 1.00  P-Amino Benzoic Acid 1.00
Py ridoxine■ HCI 1.00  Py ridoxine HCI 1.00
Ribo lavin 0.20  Ribo lavin 0.20
Thiamine■ HCI 1.00  Thiamine HCI 1.00
Cyanocobolamine 0.005  Cyanocobolamine 0.005
others g/liter  others g / liter
D— Glucose 2.00  D— Glucose 2.00
Glutathione 0.001  Glutathione 0.001
Phenol Red, Sodium 0.0053  Phenol Red, Sodium 0.0053
NaHC03 2.00 NaHC0 3 2.00
ml/liter  ml / liter
fetal bovine serum 100 pHは 7.2に調整後、 0.22um filterを用いてろ過滅菌する  fetal bovine serum 100 Adjust pH to 7.2 and sterilize by filtration using 0.22um filter
[0019] 表 2 [0019] Table 2
Amino Acids mg/liter Amino Acids mg / liter
L-Arginine 200 L-Arginine 200
L-Asparagine 50  L-Asparagine 50
L-Aspartic acid 20  L-Aspartic acid 20
l_ - Cystine ·2Η〇Ι 65 l_-Cystine2Η〇Ι 65
し一 Glutamic acid 20 Shiichi Glutamic acid 20
L-Glutamine 300  L-Glutamine 300
Glycine 10  Glycine 10
L-Histidine 15  L-Histidine 15
Hydroxy 一 l_一 proline 20  Hydroxy one l_one proline 20
L-Isoleucine 50  L-Isoleucine 50
し一し eucine 50 Eucine 50
L-Lysine-HCI 40  L-Lysine-HCI 40
し一 Methionine 15 Shiichi Methionine 15
し一 Phenylalanine 15 Shienyl Phenylalanine 15
L-Proline 20  L-Proline 20
l_一 Serine 30 l_ One Serine 30
し一 Threonine 20 Shiichi Threonine 20
L-Tryptophan 5  L-Tryptophan 5
し - Tyrosine-2Na-2H2 29 -Tyrosine-2Na-2H 2 29
0  0
l_一 Valine 20 pHは 7.2に調整後、 0.22um filterを用いてろ過滅菌する。 l_Valine 20 Adjust pH to 7.2 and sterilize by filtration using 0.22um filter.
培養後の細胞より RNAを抽出し、定量的 PCRにより TRB3の発現レベルを測定し、ァ ミノ酸飢餓での TRB3発現誘導に対する各アミノ酸の TRB3発現抑制効果を比較した 。遺伝子発現は j8 -actin発現レベルで補正した（以下記述遺伝子につ!、ても同様で ある)。 TRB3および以下に記述する遺伝子のプライマー配列を表 11, 12に示す。 データはマイナスアミノ酸培地を用いて 3時間培養後の TRB3 mRNAレベルの平均 値を 1.00として補正し表現した。  RNA was extracted from the cultured cells, the expression level of TRB3 was measured by quantitative PCR, and the TRB3 expression inhibitory effect of each amino acid on the induction of TRB3 expression by amino acid starvation was compared. Gene expression was corrected with the expression level of j8-actin (the same applies to the genes described below). Tables 11 and 12 show primer sequences of TRB3 and the genes described below. The data was expressed by correcting the mean value of TRB3 mRNA level after culturing for 3 hours using minus amino acid medium as 1.00.
その結果、ァスパラギン酸、シスチン (Cyt)、グルタミン、グリシン、ヒスチジンで有意 に TRB3発現抑制が認められた (p〈0.05) (表 3)。なお、アルギニン、ァスパラギン、ダル タミン酸、ヒドロキシプロリン、イソロイシン、ロイシン、リジン、メチォニン、フエニノレアラ ニン、プロリン、セリン、スレオニン、トリプトファン、チロシン、ノ リン、システィンについ ては有意な TRB3発現抑制は見られな力つた。  As a result, TRB3 expression was significantly inhibited by aspartic acid, cystine (Cyt), glutamine, glycine, and histidine (p <0.05) (Table 3). There is no significant inhibition of TRB3 expression for arginine, asparagine, dartamic acid, hydroxyproline, isoleucine, leucine, lysine, methionine, phenenolealanin, proline, serine, threonine, tryptophan, tyrosine, norrin, and cysteine. I helped.
また別の実験で L-ァスパラギン酸は 0.03mM〜0.3mMの添カ卩でも有意に TRB3の発 現を抑制した。ベタインは ImMの添加でも抑制効果を示さず、オル-チンは 0.3〜： Lm Mの添加では L-ァスパラギン酸と同等の抑制効果を示した力 0.03〜0.06mMでの抑 制効果は L-ァスパラギン酸より弱いものであった。 In another experiment, L-aspartic acid significantly released TRB3 even when added to 0.03 mM to 0.3 mM. Suppressed the present. Betaine does not show an inhibitory effect even when ImM is added, and ortine is 0.3- or higher. When LmM is added, the inhibitory effect is 0.03-0.06 mM, which is the same as that of L-vaspartic acid. It was weaker than acid.
表 3 培地条件 TRB3 発現レヘ レ  Table 3 Medium conditions TRB3 expression level
マイナスアミノ酸培地 1.000±0.01  Minus amino acid medium 1.000 ± 0.01
通常培地 0.179±0.07  Normal medium 0.179 ± 0.07
マイナスアミノ酸 + L-Asp 0J24±0.13  Minus amino acid + L-Asp 0J24 ± 0.13
マイナスアミノ酸 + L Cyt 0.487±0.13  Minus amino acid + L Cyt 0.487 ± 0.13
マイナスアミノ酸 + L_Gln 0J50±0.15  Minus amino acid + L_Gln 0J50 ± 0.15
マイナスアミノ酸 + L-Gly 0·665±0.12  Minus amino acid + L-Gly 0 · 665 ± 0.12
マイナスアミノ酸 + L— His 0.643±0.19 η=4平均値 ±標準偏差  Minus amino acid + L— His 0.643 ± 0.19 η = 4 Average value ± Standard deviation
〈プライマー配列〉  <Primer sequence>
配列番号 SEQIDNo： 1〜4を用いた。  SEQ ID NOs: SEQ ID Nos: 1-4 were used.
[0022] (実施例 2) (培養肝細胞に対する L-Aspのインスリン感受性増強効果） [Example 2] (Insulin sensitivity enhancing effect of L-Asp on cultured hepatocytes)
TRB3発現抑制作用を示したアミノ酸が実際に肝細胞に対するインスリン作用を増 強するか、ラット肝癌由来細胞株 Faoにおけるインスリンのグルコース- 6-ホスファタ一 ゼ (G6Pase)発現抑制作用に対する L_Asp添加の効果を検討した。  Amino acids that have shown an inhibitory effect on TRB3 expression actually enhance the insulin action on hepatocytes, or the effect of L_Asp addition on the inhibitory action of insulin on glucose-6-phosphatase (G6Pase) expression in rat liver cancer cell line Fao investigated.
Fao細胞を 24well plateに播種し 1晚培養後、 L- Aspを除いた RPMI1640培地（ダルコ ース不含、 0.1%BSA含有）を基本に 0, 0.3, l.OmM L- Aspおよび 0 , 10— ω, 10— 8, 10— 6Μ insulinを添カ卩し 6時間培養した。その後、細胞より RNAを抽出し定量的 PCRにより G6P aseの発現量測定を行った。 Fao cells were seeded on a 24-well plate, cultured for 1 cm, and then RPMI1640 medium (without dalcose, containing 0.1% BSA) excluding L-Asp was basically 0, 0.3, l.OmM L-Asp and 0, 10 - ω, 10- 8, were cultured for 6 hours添Ka卩the 10- 6 Μ insulin. Thereafter, RNA was extracted from the cells, and the expression level of G6Pase was measured by quantitative PCR.
グルコース飢餓により肝細胞の G6Paseの発現が増加する力 インスリンはこれを抑 制する。この系で L-Aspは単独添加培養でも G6Paseの発現を低下させる作用を示す とともに、インスリンと共存させることで G6Paseの発現低下を増強した。よって L-Aspは 肝細胞のインスリン感受性を増強することが確認された (表 4)。  Insulin suppresses the ability to increase G6Pase expression in hepatocytes by glucose starvation. In this system, L-Asp showed the effect of decreasing the expression of G6Pase even in the culture with addition of single, and enhanced the decrease of G6Pase expression by coexisting with insulin. Thus, L-Asp was confirmed to enhance the insulin sensitivity of hepatocytes (Table 4).
[0023] 表 4
Figure imgf000017_0001
[0023] Table 4
Figure imgf000017_0001
Insulin OM , -Asp RPMIにおける G6Pase発現レベルを 1.00とする。 The G6Pase expression level in Insulin OM, -Asp RPMI is set to 1.00.
カツコ内は各 insulin濃度で、 -Asp RPMIにおける G6Pase発現レベルを 1.00とした場 合の値。  The values in Katsuko are for each insulin concentration, and the G6Pase expression level at -Asp RPMI is 1.00.
〈プライマー配列〉  <Primer sequence>
配列番号 SEQIDNo : l , 2, 5, 6を用いた。  SEQ ID NO: SEQ ID No: 1, 2, 5, 6 was used.
[0024] (実施例 3) (病態ラットに対する L-Asp長期投与での肝遺伝子発現変動及びインスリ ン感受性増強効果） [Example 3] (Liver gene expression change and insulin sensitivity enhancement effect by long-term administration of L-Asp to diseased rats)
in vitroの培養細胞株で見られた L-Aspのインスリン感受性増強作用が in vivoの病 態動物で十分な効果を示すかを、インスリン分泌不全 Zインスリン作用不足の糖尿 病モデルラットである GKラットに L-Aspを混餌で長期投与し、肝遺伝子発現並びに病 態に対する効果を検討した。  The effect of L-Asp on the sensitivity of insulin observed in cultured cell lines in vitro shows sufficient effects in in vivo disease animals. GK rat, a diabetic model rat with insufficient insulin secretion Z In addition, L-Asp was administered for a long time with diet, and the effect on liver gene expression and disease state was examined.
雄性 GKラットを 6週齢で購入し、 1週間予備飼育後 1群 5匹で下記の群に分け、実験 食に切り替え、 8週間飼育した。  Male GK rats were purchased at 6 weeks of age, and after 1 week pre-breeding, 1 group was divided into 5 groups per group, switched to the experimental diet and bred for 8 weeks.
CON群：コントロール食投与  CON group: Control diet
3AS群： 3% L-Asp含有食投与  3AS group: 3% L-Asp containing diet
6AS群： 6% L-Asp含有食投与  6AS group: 6% L-Asp containing diet
[0025] 投与 6週目における絶食時血糖は、 L-Asp投与量に依存して低下し、 6AS群では C ON群に比べて有意に低値を示した。また絶食時インスリンレベルは CON群に比べて 3AS群で有意に低値を示し、 6AS群で低下傾向を示した。これらより L- Asp投与〖こより GKラットにおけるインスリン抵抗性が改善する事が考えられた。絶食時血糖は一般に インスリン基礎分泌並びにインスリン抵抗性を反映する指標と考えられており、長期 の Asp投与力 Sインスリン基礎分泌の改善及び/または肝遺伝子発現を反映したイン スリン感受性の増強作用を示し、血糖値の低下を促進したと考えられた。 投与 8週目で 18時間絶食後に肝臓を摘出し、 RNAを抽出して定量的 PCRにより遺 伝子発現を比較した。その結果、インスリンシグナルを抑制する TRB3の発現は 3AS 群で CON群に比べて有意に低下し 6AS群でも低下傾向が認められた。また G6Paseも 6AS群で有意な低下が認められた。さらに LARの発現力 ¾AS, 6AS群で低下傾向を示 した (表 5)。 [0025] Fasting blood glucose at the 6th week after administration decreased depending on the dose of L-Asp, and the 6AS group showed a significantly lower value than the CON group. Fasting insulin levels were significantly lower in the 3AS group than in the CON group and decreased in the 6AS group. From these results, it was considered that insulin resistance in GK rats was improved by L-Asp administration. Fasting blood glucose is generally considered to be an index that reflects basal insulin secretion and insulin resistance, and it has a long-term ability to administer Asp. It shows an improvement in insulin basal secretion and / or an increase in insulin sensitivity that reflects liver gene expression. It was thought that it promoted a decrease in blood sugar level. At 8 weeks after administration, the liver was removed after 18 hours of fasting, RNA was extracted, and gene expression was compared by quantitative PCR. As a result, the expression of TRB3, which suppresses insulin signal, was significantly lower in the 3AS group than in the CON group, and decreased in the 6AS group. G6Pase also decreased significantly in the 6AS group. Furthermore, the LAR expression ability ¾AS and 6AS groups showed a decreasing trend (Table 5).
以上より、 L-Aspは病態動物への長期投与において、 in vitro同様、遺伝子発現を修 飾し、肝臓のインスリン感受性を増強することが想定された。  Based on the above, it was postulated that L-Asp modifies gene expression and enhances insulin sensitivity in the liver, as in vitro, for long-term administration to diseased animals.
[0026] 表 5 [0026] Table 5
投与（過） CON 3AS 6AS  Administration (excess) CON 3AS 6AS
6weeks 空腹時血糖（mg/dl) 6 152±3.6 151 ±4.8 140±8.9* 6weeks Fasting blood glucose (mg / dl) 6 152 ± 3.6 151 ± 4.8 140 ± 8.9 *
6weeks 空腹時インスリン（ng/ml) 6 2.5±0.6 1.7±0.4* 2.0±0.26weeks Fasting insulin (ng / ml) 6 2.5 ± 0.6 1.7 ± 0.4 * 2.0 ± 0.2
8weeks TRB3 8 1.00±0.41 0.49±0.1 0.61 ±0.198weeks TRB3 8 1.00 ± 0.41 0.49 ± 0.1 0.61 ± 0.19
8weeks G6Pase 8 1.00±0.20 0.99±0.15 0.74±0.06*8weeks G6Pase 8 1.00 ± 0.20 0.99 ± 0.15 0.74 ± 0.06 *
8weeks LAR 8 1.00±0.20 0.70±0.15 0.68±0.18 8weeks LAR 8 1.00 ± 0.20 0.70 ± 0.15 0.68 ± 0.18
[0027] η=5平均値士標準偏差 [0027] η = 5 mean value standard deviation
mRNAレベルは CON群のレベルを 1.00とする  mRNA level is 1.00 for CON group
*： t- testにより CON群に比べて有意差あり (P〈0.05)  *: Significantly different from CON group by t-test (P 〈0.05)
〈プライマー配列〉  <Primer sequence>
配列番号 SEQIDNo： 1〜8を用いた。  SEQ ID NOs: SEQ ID Nos: 1-8 were used.
また各群の個体の肝遺伝子を等量ずつ混合した試料を用いて遺伝子発現プロファ ィリングを行った。遺伝子発現プロフアイリングには Gene Chipラットゲノム U34Aアレイ ( Alfymetrix社)を用いた。その結果、脂肪合成に関与する malic enzyme(ME), stearoyl -CoenzymeA desaturase 1 (SCD1)遺伝子の肝での発現低下が見られたことから、 L- Aspの長期投与は肝脂質合成を抑制することも想定された (表 6)。  In addition, gene expression profiling was performed using samples in which equal amounts of liver genes from individuals in each group were mixed. Gene Chip rat genome U34A array (Alfymetrix) was used for gene expression profiling. As a result, the expression of malic enzyme (ME) and stearoyl-CoenzymeA desaturase 1 (SCD1) genes involved in fat synthesis was decreased in the liver, and long-term administration of L-Asp suppressed liver lipid synthesis. (Table 6).
[0028] 表 6 [0028] Table 6
投与（週） C0N 3 AS 6 AS  Administration (week) C0N 3 AS 6 AS
8weeks ME 8 1.00 0.55 0.62  8weeks ME 8 1.00 0.55 0.62
8weeks SCD1 8 1.00 0.78 0.71 n=5平均値 mRNAレベルは CON群のレベルを 1.00とする 8weeks SCD1 8 1.00 0.78 0.71 n = 5 average mRNA level is 1.00 for CON group
(実施例 4) (病態ラットに対する L-Asp長期投与でのインスリン抵抗性改善作用及び 脂肪肝改善作用） (Example 4) (Insulin resistance improving effect and fatty liver improving effect by long-term administration of L-Asp on diseased rats)
近年のインスリン抵抗性に関連する代謝異常疾患、例えば糖尿病や脂肪肝の増加 には脂肪摂取量の増加が大きな要因になっているとされていることから、高脂肪食を 与えた GKラットを用いて L-Aspの薬理作用を確認した。  GK rats fed a high-fat diet are considered to be a major factor in the metabolic disorders related to insulin resistance in recent years, such as diabetes and increased fatty liver. The pharmacological action of L-Asp was confirmed.
表 7に示すような組成で通常食 (5%ダイズ油を含む；以下 SDと略す）、高脂肪食 (30 %牛脂を含む；以下 HFDと略す)及び HFDに L-Aspを約 6%添加した実験食を作成し た。さらに陽性対照として HFDに 0.01%ピオダリタゾン (Pio)を添加した実験食を作成 した。  Add about 6% L-Asp to normal diet (including 5% soybean oil; hereinafter abbreviated as SD), high fat diet (including 30% beef tallow; hereinafter abbreviated as HFD) and HFD with the composition shown in Table 7. An experimental food was prepared. As a positive control, an experimental meal was prepared by adding 0.01% piodaritazone (Pio) to HFD.
表 7 SD群 HFD群 HFD+ASD群 HFD+ Table 7 SD group HFD group HFD + ASD group HFD +
コーンスターチ 5175 2675 2675 2675 Cornstarch 5175 2675 2675 2675
αスターチ 1320 1320 1320 1320 Alpha starch 1320 1320 1320 1320
大豆油 500 Soybean oil 500
牛脂 3000 3000 3000 Tallow 3000 3000 3000
カゼイン 2000 2000 2000 2000 Casein 2000 2000 2000 2000
L-ァスパラギン酸 600  L-aspartic acid 600
セルロース 500 500 500 500 Cellulose 500 500 500 500
salt mix(AIN93G) 350 350 350 350 salt mix (AIN93G) 350 350 350 350
vitamin mix(AIN93) 100 100 100 100 vitamin mix (AIN93) 100 100 100 100
シスチン 30 30 30 30 Cystine 30 30 30 30
choline bitartrate 25 25 25 25 choline bitartrate 25 25 25 25
Tert-butylhydroquinone 0.14 0.14 0.14 0.14  Tert-butylhydroquinone 0.14 0.14 0.14 0.14
ピ才グリタゾン 1 Pi years old gritazone 1
(g/10kg diet)  (g / 10kg diet)
7週令の雄性 GKラットを 2週間通常食で飼育後 1群 6匹に群分けを行った。 HFD群 では、 SD群に比し空腹時血糖並びに空腹時血漿インスリンレベルの増加が認められ 、インスリン抵抗性の存在が示唆された。これに対し HFD+Asp群ではこの上昇が抑制 されており、 L-Aspにインスリン抵抗性の改善効果が認められた (表 8)。その作用は 陽性対照としたピオグリタゾンとほぼ同等であった。 Seven-week-old male GK rats were bred on a normal diet for 2 weeks and then divided into 6 groups per group. In the HFD group, fasting blood glucose and fasting plasma insulin levels were increased compared to the SD group, suggesting the presence of insulin resistance. In contrast, this increase was suppressed in the HFD + Asp group. L-Asp was found to improve insulin resistance (Table 8). The effect was almost the same as pioglitazone as a positive control.
また空腹時血漿トリグリセリドについては SD群に比し HFD群で高値を示すことはな かったが、 HFD+Asp群ではこれも低下した（表 8)。  Fasting plasma triglycerides were not higher in the HFD group than in the SD group, but decreased in the HFD + Asp group (Table 8).
次に定法に従い肝臓中のトリグリセリド含量を測定したところ、 HFD群では肝臓 lg当 たり 39.9mgと、 SD群（17.1mg)に比し、肝臓中の脂肪含量の増加が認められた。これ に対し HFD+Asp群では、肝臓中のトリグリセリドは肝臓 lg当たり 28.0mgと低下しており 、その効果はピオグリタゾンより強いものであった (表 8)。また組織学的な検討でも肝 門脈域を中心とした脂肪滴蓄積の改善が認められ、 L-Aspの脂肪肝、 NASHに対す る有効性が示された。  Next, when the triglyceride content in the liver was measured according to a conventional method, the fat content in the liver was found to be 39.9 mg per lg in the HFD group compared to the SD group (17.1 mg). In contrast, in the HFD + Asp group, the triglyceride in the liver decreased to 28.0 mg / lg of liver, and the effect was stronger than that of pioglitazone (Table 8). Histological examination also showed improvement in lipid droplet accumulation mainly in the hepatic portal vein area, indicating the effectiveness of L-Asp for fatty liver and NASH.
なお本試験を通じ、 HFD+Pio群では HFD群に比し有意な体重増加が認められたが 、 HFD+Asp投与群の体重推移は HFD群と同等であった (表 8)。  Throughout this study, the HFD + Pio group showed a significant increase in body weight compared to the HFD group, but the HFD + Asp-administered group had the same body weight transition as the HFD group (Table 8).
また血液生化学検査 (ALT、 AST)、及び組織学的検査において、全群で肝機能障 害は認められなかった。  In the blood biochemical tests (ALT, AST) and histological examination, no liver dysfunction was observed in all groups.
[0031] 表 8 [0031] Table 8
Figure imgf000020_0001
η=6平均士標準偏差
Figure imgf000020_0001
η = 6 average standard deviation
[0032] さらに 6週投与後のラット肝臓中の遺伝子発現を定量的 PCRにより調べたところ、 Η FD群に比し HFD+Asp群では TRB3、 G6Paseの発現はそれぞれ 0.58倍、 0.52倍に低 下しており、これまでの結果同様肝インスリン感受性の改善を示す遺伝子発現の変 動が確認された。 [0032] Further, the quantitative expression of gene expression in the rat liver after 6 weeks of administration was examined. Compared to the FD group, TRB3 and G6Pase expression decreased 0.58 and 0.52 times in the HFD + Asp group, respectively, and the changes in gene expression showing improved liver insulin sensitivity were confirmed. .
また PPAR a、 ACO、 CPT-1の発現はそれぞれ 1.34倍、 1.18倍、 1.20倍に亢進して おり、 L-Asp投与は肝での脂肪燃焼に関わる酵素の発現を転写因子レベルで促進 することも示唆された。  In addition, the expression of PPARa, ACO, and CPT-1 increased by 1.34, 1.18, and 1.20, respectively, and L-Asp administration promotes the expression of enzymes involved in fat burning in the liver at the transcription factor level. Also suggested.
[0033] 表 9 [0033] Table 9
Figure imgf000021_0001
n=6平均値
Figure imgf000021_0001
n = 6 average
mRNAレベルは SD群のレベルを 1.00とする  mRNA level is 1.00 for the SD group
〈プライマー配列〉  <Primer sequence>
配列番号 SEQIDNo : l〜6 ,9〜14を用いた。  SEQ ID NOs: SEQ ID Nos: 1 to 6 and 9 to 14 were used.
これらの結果より我々が見出した L-Aspのインスリン感受性亢進、糖新生抑制作用 は、 in vitroのみならず、インスリン分泌不全とインスリン抵抗性を併せ持つ in vivo病 態モデルでも効果を発揮すること、同時に高脂肪食摂取による脂肪肝も改善する効 果を示すことが確かめられた。  From these results, we found that L-Asp enhances insulin sensitivity and suppresses gluconeogenesis not only in vitro but also in in vivo pathological models that combine insulin secretion failure and insulin resistance. It was confirmed that the effect of improving fatty liver by ingesting a high-fat diet showed an effect.
[0034] (実施例 5) (L-Aspのインスリン抵抗性関連遺伝子 (TRB3)発現抑制および肝脂質代 謝関連遺伝子 (HNF4 a )発現亢進に対する他アミノ酸添加の効果 (Example 5) (Inhibition of L-Asp Insulin Resistance Related Gene (TRB3) Expression and Effect of Addition of Other Amino Acids on Hepatic Lipid Metabolism Related Gene (HNF4 a) Expression Enhancement)
L-Aspのラット肝細胞由来細胞株 Faoの TRB3発現に対する抑制効果が他のアミノ 酸として L-Alaをカ卩えることで増強されるかを調べた。 また同時に HNF4 aの発現が 亢進されるかを調べた。  We investigated whether the inhibitory effect of L-Asp on the TRB3 expression of the rat hepatocyte-derived cell line Fao can be enhanced by adding L-Ala as another amino acid. At the same time, it was examined whether the expression of HNF4a was enhanced.
Fao細胞 2x 105cells/wellを 24well plateに播き、 10%牛胎児血清含有 RPMI 1640培 地（ナカライテスタ株式会社 Code No.30264-85 )で 37°C、 C02 5%条件下 1晚培養し た。翌日、アミノ酸を全て除いた RPMI培地（マイナスアミノ酸培地、表 1)、またはこれ に L-Asp (濃度 0.05mM)あるいは L-Ala (濃度 0.03mM)、または両者を添カ卩した培地 に交換し、 37°C、 C02 5%条件下でさらに 3時間培養した。培養後、細胞から RNAを抽 出し、定量的 PCRにより TRB3 mRNAおよび HNF4 a mRNAのレベルを測定した（表 1 0)。各 mRNA発現量は、マイナスアミノ酸培地でのレベルを 1.00とした相対値で示し た。 Fao cells (2 × 105 cells / well) were seeded on a 24-well plate and cultured for 1% at 37 ° C. and C02 5% in RPMI 1640 medium (Nacalai Tester Code No. 30264-85) containing 10% fetal calf serum. The next day, RPMI medium (minus amino acid medium, Table 1) with all amino acids removed, or this The medium was replaced with a medium supplemented with L-Asp (concentration 0.05 mM) or L-Ala (concentration 0.03 mM), or both, and the cells were further cultured at 37 ° C, C02 5% for 3 hours. After culture, RNA was extracted from the cells, and the levels of TRB3 mRNA and HNF4a mRNA were measured by quantitative PCR (Table 10). Each mRNA expression level was expressed as a relative value with the level in the minus amino acid medium being 1.00.
表 10 Table 10
Figure imgf000022_0001
Figure imgf000022_0001
TRB3 1.00 0.54 0.43 0.26  TRB3 1.00 0.54 0.43 0.26
HNF4 a 1.00 1.30 0.80 1.84  HNF4 a 1.00 1.30 0.80 1.84
[0035] TRB3 mRNAの発現レベルは、培地に L-Aspあるいは L-Alaを添カ卩したとき 54%ある いは 43%まで抑制され、 L-Aspおよび L-Ala両者を添カ卩したときにはより強 、発現抑 制（26%)が認められた（26%)。  [0035] The expression level of TRB3 mRNA was suppressed to 54% or 43% when L-Asp or L-Ala was added to the medium, and when both L-Asp and L-Ala were added. More strongly, suppression of expression (26%) was observed (26%).
また HNF4 a mRNAの発現レベルは L-Alaを添カ卩したときには上昇は認められなか つたが、 L-Asp添カ卩では 1.3倍まで上昇し、さらに L-Aspおよび L-Ala両者を添カ卩した ときには L-Asp単独よりもはるかに強 、発現上昇（ 1.84倍）が認められた。  In addition, the expression level of HNF4 a mRNA did not increase when L-Ala was added, but increased to 1.3-fold when L-Asp was added, and both L-Asp and L-Ala were added. When drunk, it was much stronger than L-Asp alone, with an increased expression (1.84 times).
<プライマー配列 >  <Primer sequence>
配列番号 SEQIDNo. 3、 4、 15及び 16を用いた。  SEQ ID NOs: SEQ ID Nos. 3, 4, 15 and 16 were used.
遺伝子発現測定に用いたプライマー配列を表 11、表 12に示す。  The primer sequences used for gene expression measurement are shown in Tables 11 and 12.
[0036] 表 11 [0036] Table 11
pr i mer name pr imer name SEQ ID NO sequence pr i mer name pr imer name SEQ ID NO sequence
Rat iS-Acti n-3442F β -Act n F SEQ ID NO 1 CTCCAAGTATCCACGGCATAG Rat iS-Acti n-3442F β -Act n F SEQ ID NO 1 CTCCAAGTATCCACGGCATAG
Rat iS-Acti β -Act n R SEQ ID NO 2 AAGCAATGCTGTCACCTTCC rTRB3 F TRB3 F SEQ ID NO 3 GTTACCACAGTGCCACATGC rTRB3 TRB3 R SEQ ID NO 4 CAGAAGCCTAGCACCAGACCRat iS-Acti β -Act n R SEQ ID NO 2 AAGCAATGCTGTCACCTTCC rTRB3 F TRB3 F SEQ ID NO 3 GTTACCACAGTGCCACATGC rTRB3 TRB3 R SEQ ID NO 4 CAGAAGCCTAGCACCAGACC
Rat G6Pase- 1998F G6Pase F SEQ ID NO 5 ACACACTCTGCAAACCAGCCRat G6Pase- 1998F G6Pase F SEQ ID NO 5 ACACACTCTGCAAACCAGCC
Rat G6Pase- 2116R G6Pase R SEQ ID NO 6 CCTGGAGCAGAAATGACTCC rLAR-F o LA F SEQ ID NO 7 CTCTGAGCCATACCGACCATC rLAR- LA R SEQ ID NO 8 CAATGTTCCCGAAATGCTGTGRat G6Pase- 2116R G6Pase R SEQ ID NO 6 CCTGGAGCAGAAATGACTCC rLAR-F o LA F SEQ ID NO 7 CTCTGAGCCATACCGACCATC rLAR- LA R SEQ ID NO 8 CAATGTTCCCGAAATGCTGTG
PPARaF PPAR F SEQ ID NO 9 CTCGTGCAGGTCATCAAGAAPPARaF PPAR F SEQ ID NO 9 CTCGTGCAGGTCATCAAGAA
PPARa PPA R SEQ ID NO 10 CAGCCCTCTTCATCTCCAAGPPARa PPA R SEQ ID NO 10 CAGCCCTCTTCATCTCCAAG
ACOF ACO F SEQ ID NO 11 GAAGTTGGTG6GTGGTATGGACOF ACO F SEQ ID NO 11 GAAGTTGGTG6GTGGTATGG
ACOR ACO R SEQ ID NO 12 CGAACAAGGTCGACAGAGGT rCPT1a-L CPT1 F SEQ ID NO 13 TATTTTCCAGTGCCCCTTTG rCPT1a- CPT1 R SEQ ID NO U ACTGTTCAGCCAGCACCTCT ACOR ACO R SEQ ID NO 12 CGAACAAGGTCGACAGAGGT rCPT1a-L CPT1 F SEQ ID NO 13 TATTTTCCAGTGCCCCTTTG rCPT1a- CPT1 R SEQ ID NO U ACTGTTCAGCCAGCACCTCT
[0037] 表 12 [0037] Table 12
Primer name primer name SEQ ID NO sequence  Primer name primer name SEQ ID NO sequence
HNF4 a -F HNF4 a ~F SEQ ID NO: 15 CATAGTTGCCAACACGATGC HNF4 a -R HNF4 a -R SEQ ID NO: 16 TGGCAGGAGCTTGTAGGATT HNF4 a -F HNF4 a ~ F SEQ ID NO: 15 CATAGTTGCCAACACGATGC HNF4 a -R HNF4 a -R SEQ ID NO: 16 TGGCAGGAGCTTGTAGGATT
[0038] (実施例 6) (正常ラット-高脂肪食負荷モデルに対する L-Asp長期投与での脂肪肝改 善作用） [0038] (Example 6) (Normal rat-high-fat diet load model, L-Asp long-term administration improves fatty liver)
高脂肪食を与えた Wistarラットを用いて L-Aspの薬理作用を確認した。表 7に示すよ うな組成で高脂肪食 (30%牛脂を含む；以下 HFDと略す)及び HFDに L-Aspを約 6% 添加した実験食を作成した。  The pharmacological action of L-Asp was confirmed using Wistar rats fed a high fat diet. A high fat diet (containing 30% beef tallow; hereinafter abbreviated as HFD) and an experimental diet with about 6% L-Asp added to HFD were prepared as shown in Table 7.
7週令の雄性 Wistarラットを 2週間通常食 (CRF-1)で飼育後 1群 6匹に群分けを行つ た。 4週間実験食を摂餌させたところ、 HFD群に対し HFD+Asp群では体重が低下傾 向にあり、 L-Aspに体重抑制作用が認められた (表 13)。  Seven-week-old male Wistar rats were bred on a normal diet (CRF-1) for 2 weeks and then divided into 6 groups per group. When the experimental diet was fed for 4 weeks, the body weight decreased in the HFD + Asp group compared to the HFD group, and L-Asp had a body weight-inhibitory effect (Table 13).
次に定法に従い肝臓中のトリグリセリド含量を測定したところ、 HFD群では肝臓 lg当 たり 57.6mgを示したのに対し、 HFD+Asp群では、肝臓中のトリグリセリドは肝臓 lg当 たり 43.4mgと低下しており、 L-Aspの肝 TG蓄積抑制作用が認められた（表 13)。また 全肝臓当たりに換算しても、 HFD群では全肝臓当たり 1047.5mgを示したのに対し、 H FD+Asp群では 743.4mgと低下しており、同様の作用が認められた (表 13)。以上の結 果から、 L-Aspは高脂肪食による体重増加や肝の脂肪化を予防'軽減する有効性が 示された。 Next, when the triglyceride content in the liver was measured according to the standard method, it showed 57.6 mg / lg of liver in the HFD group, whereas in the HFD + Asp group, triglyceride in the liver decreased to 43.4 mg / lg of liver. L-Asp was found to suppress hepatic TG accumulation (Table 13). In terms of total liver, the HFD group showed 1047.5 mg per whole liver, whereas the H FD + Asp group showed a decrease of 743.4 mg, which was similar (Table 13). . From the above results, L-Asp is effective in preventing and reducing weight gain and hepatic steatosis due to a high-fat diet. Indicated.
表 13Table 13
Figure imgf000024_0001
Figure imgf000024_0001
n=6平均士標準偏差  n = 6 average standard deviation

Claims

請求の範囲 The scope of the claims
[1] インスリン感受性増強作用を有するアミノ酸を有効成分とする肝疾患予防及び Zま たは治療用組成物。  [1] A composition for preventing and / or treating liver disease comprising an amino acid having an insulin sensitivity enhancing action as an active ingredient.
[2] 肝疾患が、インスリン抵抗性関連肝疾患である請求項 1記載の肝疾患予防及び Z または治療用組成物。  [2] The composition for preventing and / or treating liver disease according to claim 1, wherein the liver disease is insulin resistance-related liver disease.
[3] インスリン抵抗性関連肝疾患が非アルコール性脂肪肝もしくは非アルコール性脂 肪性肝炎である請求項 2記載の肝疾患予防及び Zまたは治療用組成物。  [3] The composition for preventing and / or treating liver disease according to claim 2, wherein the insulin resistance-related liver disease is non-alcoholic fatty liver or non-alcoholic fatty hepatitis.
[4] インスリン感受性増強作用を有するアミノ酸が、ァスパラギン酸、了ラニン、シスチン 、グルタミン、グリシン、ヒスチジンおよびそれらの塩力もなる群力も選ばれる少なくとも 1つを含むものである請求項 1〜3いずれか 1項記載の肝疾患予防及び Zまたは治 療用組成物。  [4] The amino acid having an insulin sensitivity enhancing action includes at least one selected from aspartic acid, rylanin, cystine, glutamine, glycine, histidine and a group power that also has a salt power thereof. The composition for prevention and Z or treatment of liver diseases as described.
[5] インスリン感受性増強作用を有するアミノ酸が、 L-ァスパラギン酸またはその塩であ る請求項 1〜4のいずれ力 1項記載の肝疾患予防及び Zまたは治療用組成物。  5. The composition for preventing and / or treating liver disease according to any one of claims 1 to 4, wherein the amino acid having an insulin sensitivity enhancing action is L-aspartic acid or a salt thereof.
[6] L-ァスパラギン酸および Zまたはその塩、および第二の有効成分としてァラニン、 シスチン、グルタミン、グリシン、ヒスチジンおよびそれらの塩からなる群力も選ばれる 少なくとも 1つのアミノ酸を有効成分として含有することを特徴とする肝疾患予防及び Zまたは治療用組成物。  [6] L-aspartic acid and Z or a salt thereof, and a group force consisting of alanine, cystine, glutamine, glycine, histidine and a salt thereof as a second active ingredient is also selected As an active ingredient A composition for preventing and / or treating liver diseases characterized by the following.
[7] L-ァスパラギン酸および Zまたはその塩、および第二の有効成分としてァラニンま たはその塩を有効成分として含有することを特徴とする肝疾患予防及び Zまたは治 療用組成物。  [7] A composition for preventing and treating liver disease or Z, comprising L-aspartic acid and Z or a salt thereof, and alanan or a salt thereof as an active ingredient as an active ingredient.
[8] L-ァスパラギン酸および Zまたはその塩、および第二の有効成分としてアミノ酸及 びその塩以外の肝疾患予防'治療薬を含有する肝疾患予防及び Zまたは治療用組 成物。  [8] A composition for the prevention and Z or treatment of liver disease comprising L-aspartic acid and Z or a salt thereof, and as a second active ingredient a therapeutic agent for liver disease prevention other than amino acids and salts thereof.
[9] L-ァスパラギン酸および Ζまたはその塩を単独の有効成分とする組成物と、請求 項 6〜8記載の第二の有効成分を含有してなる組成物の少なくとも一つとを組み合わ せたことを特徴とする肝疾患予防及び Ζまたは治療用キット。  [9] A composition containing L-aspartic acid and cocoon or a salt thereof as a single active ingredient, and at least one of the compositions containing the second active ingredient according to claim 6-8. A liver disease prevention and epilepsy or treatment kit characterized by the above.
[10] インスリン感受性増強作用を有するアミノ酸を有効成分とするインスリン抵抗性関連 疾患予防及び Ζまたは治療用組成物。 [10] A composition for preventing and / or treating insulin resistance-related diseases, comprising an amino acid having an insulin sensitivity enhancing action as an active ingredient.
[11] インスリン抵抗性関連疾患力 メタボリックシンドロームもしくは代謝症候群関連疾患 である請求項 10記載のインスリン抵抗性関連疾患予防及び Zまたは治療用組成物 [11] The composition for preventing and / or treating insulin resistance-related diseases according to claim 10, which is a disease related to insulin resistance or a metabolic syndrome-related disease.
[12] インスリン感受性増強作用を有するアミノ酸が、ァスパラギン酸、了ラニン、シスチン[12] Amino acids having an insulin-sensitizing effect are aspartic acid, Ranan, cystine
、グルタミン、グリシン、ヒスチジンおよびそれらの塩力もなる群力も選ばれる少なくとも, Glutamine, glycine, histidine and their salt strength are also selected at least
1つを含むものである請求項 10又は 11記載のインスリン抵抗性関連疾患予防及びInsulin resistance-related disease prevention according to claim 10 or 11, wherein
Zまたは治療用組成物。 Z or therapeutic composition.
[13] インスリン感受性増強作用を有するアミノ酸が、 L-ァスパラギン酸および Zまたはそ の塩である請求項 10〜12のいずれか 1項記載のインスリン抵抗性関連疾患予防及 び Zまたは治療用組成物。  [13] The composition for preventing and / or treating insulin resistance-related diseases according to any one of claims 10 to 12, wherein the amino acid having an insulin sensitivity enhancing action is L-aspartic acid and Z or a salt thereof. .
[14] 請求項 1〜13の 、ずれか 1項記載の予防及び Zまたは治療用組成物を含有する  [14] The preventive and Z or therapeutic composition according to any one of claims 1 to 13,
TOo  TOo
[15] L-ァスパラギン酸またはその塩を有効成分とする肝疾患予防及び Zまたは治療用 組成物を含有する機能性食品。  [15] A functional food containing a composition for preventing and treating liver disease or treatment comprising L-aspartic acid or a salt thereof as an active ingredient.
[16] L-ァスパラギン酸またはその塩を有効成分とするインスリン抵抗性関連疾患予防及 び Zまたは治療用組成物を含有する機能性食品。 [16] A functional food containing a composition for preventing and / or treating insulin resistance-related diseases comprising L-aspartic acid or a salt thereof as an active ingredient.
[17] ァスパラギン酸、ァラニン、シスチン、グルタミン、グリシン、ヒスチジンおよびそれら の塩カゝらなる群カゝら選ばれる少なくとも 1つを含むインスリン感受性増強剤。 [17] An insulin sensitivity enhancer comprising at least one selected from the group consisting of aspartic acid, alanine, cystine, glutamine, glycine, histidine and salts thereof.
[18] ァスパラギン酸、ァラニン、シスチン、グルタミン、グリシン、ヒスチジンおよびそれら の塩力 なる群力 選ばれる少なくとも 1つを含むインスリン抵抗性改善剤。 [18] An insulin resistance ameliorating agent comprising at least one selected from aspartic acid, alanine, cystine, glutamine, glycine, histidine and a group power of salt power thereof.
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